Bioactive Soft Tissue Implant and Methods of Manufacture and Use Thereof

ABSTRACT

A bioactive filamentary structure includes a sheath coated with a mixture of synthetic bone graft particles and a polymer solution forming a scaffold structure. In forming such a structure, synthetic bone graft particles and a polymer solution are applied around a filamentary structure. A polymer is precipitated from the polymer solution such that the synthetic bone graft particles and the polymer coat the filamentary structure and the polymer is adhered to the synthetic bone graft particles to retain the graft particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 15/584,620 filed May 2, 2017, now U.S. Pat. No.10,729,548, which claims the benefit of the filing date of U.S.Provisional Patent Application No. 62/330,584 filed May 2, 2016, and isa continuation-in-part of U.S. patent application Ser. No. 15/234,239filed Aug. 11, 2016, published as U.S. Patent Application PublicationNo. 2017/0043052 A1, which claims the benefit of the filing date of U.S.Provisional Patent Application No. 62/204,119, filed Aug. 12, 2015, thedisclosures of all of which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to bone suture anchors, and moreparticularly, to bioactive suture anchors and the preparation thereof.

BACKGROUND OF THE INVENTION

Surgical sutures and suture anchors are used to close or hold togethervarious types of soft tissue, including but not limited to skin, bloodvessels, and internal organ tissue. In many instances, such sutures aswell as all-suture suture anchors are made of biocompatible materialssuch as but not limited to non-absorbable materials such as cellulose(cotton, linen), protein (silk), processed collagen, nylon, polyester,polypropylene, aromatic polyamides (“aramid”), polytetraflourethylene,steel, copper, silver, aluminum, various alloys and the like, includingmany proprietary polymers and composites, to bioabsorbable (orbiodegradable or bioerodible) synthetic materials, such as polymers andcopolymers of glycolic and lactic acid. In some instances, such sutureshave been coated with materials that provide additional benefitsincluding antimicrobial, tribological properties, further biocompatibleproperties, and as well as materials that have properties to promotetissue growth and repair, including biodegradable matrices of growthfactor molecules.

These suture and coating combinations often do not satisfy their desiredefficacy as coatings are either removed during insertion of coatedsutures due to abrasive contact with neighboring tissue or are rapidlydegraded and diffused into the body before the coating can exertsignificant beneficial effects at the target site. Supplying highconcentrations of bioactive material to the target site is not aneffective solution as such concentrations may negate the benefits andindeed could be harmful, and further are relatively costly.

Attempting to address these issues, complex suture designs have utilizeda multifilament biodegradable porous core surrounded by a biodegradablebraided or woven sheath in which a concentration of living cells areretained within interstices disposed between the filaments of the core.In such designs, the sheath inhibits migration of the living cells fromthe porous core.

These designs require a concentration of bioactive material such asliving cells and therapeutic agents throughout the cores and thus cannotbe used with standard sutures as cores. Accordingly, other methods ofpreparation are needed to provide biocompatibility and tissue growthpromotion benefits to surgical sutures and anchors made of suturematerial.

Bone graft materials are used in many applications within the orthopedicand/or other medical fields, such as to cause fusion of adjacent boneparts to repair a fracture, to fuse a joint(s) and alleviate pain at thejoint(s), and/or more securely attach an implant or other device tobone. Bone graft materials have numerous indications within theorthopedic field, which rely on the ability of the material tofacilitate natural bone growth at the repair site (e.g., forregenerating and/or forming additional bone at the site).

There has been a continuing need for improved bone graft materials. Forexample, autograft devices, which are processed from a patient's ownbone, have the ideal properties and radiopacity. However, the use ofautogenous bone exposes the patient to a second surgery, pain, andmorbidity at the donor site. Allograft devices, which are processed fromdonor bone, also carry the risk of disease transmission. These devicesare restricted in terms of variations on shape and size and havesub-optimal strength properties that decrease after implantation. Thequality of the allograft devices varies because the devices are natural.Also, since companies that provide allograft implants obtain theirsupply from donor tissue banks, there tend to be limitations on supply.

In recent years, synthetic materials have become a viable alternative toautograft and allograft devices. Most synthetic materials share numerousadvantages over allografts, autografts and demineralized bone matrix(“DBM”), such as unlimited supply, elimination of disease transmission,elimination of second surgery, and the ability to be shaped into variousshapes and sizes. Many synthetic bone grafts include materials thatclosely mimic mammalian bone, such as compositions containing calciumphosphates. Exemplary calcium phosphate compositions contain type-Bcarbonated hydroxyapatite [Ca₅(PO₄)_(3x)(CO₃)x(OH)], which is theprincipal mineral phase found in the mammalian body. Calcium phosphateceramics have been fabricated and implanted in mammals in various formsincluding, but not limited to, shaped bodies and cements. Differentstoichiometric compositions such as hydroxyapatite (“HAp”), tricalciumphosphate (“TCP”), tetracalcium phosphate (“TTCP”), and other calciumphosphate salts and minerals, have all been employed to match theadaptability, biocompatibility, structure, and strength of natural bone.The role of pore size and porosity profile in promotingrevascularization, healing, and remodeling of bone have been recognizedas a critical property for bone grafting materials. The preparation ofexemplary porous calcium phosphate materials that closely resemble bonehave been disclosed, for instance, in U.S. Pat. Nos. 6,383,519;6,521,246 and 6,991,803, which are incorporated herein by reference intheir entirety.

The Vitoss® line of synthetic bone graft products (manufactured byStryker Orthobiologics, Malvern, Pa.) includes β-TCP (i.e., betatri-calcium phosphate), collagen, and/or bioactive glass.

Scaffolds may be used to support bone graft materials. Among the mostcommonly used scaffolds in medical devices are mammalian-derivedcollagens (A. Oyran et al., J. Orthop. Surg. Res. 2014 Mar. 17;9(1):18).

Synthetic scaffolds using biocompatible polymers, such as PCL are alsoavailable and may be formed by, or using, molding, solvent casting,particulate leaching, solvent evaporation, electrospun fibers or meshesthereof, and 3-D printed constructs. (see M. A. Woodruff and D. W.Hutmacher, The return of a forgotten polymer: Polycaprolactone in the21^(st) century. Progress in Polymer Science, p. 1-102, Elsevier Press(2010)) Highly ordered, crystalline constructs having properties akin tothose of solvent casted scaffolds can also be formed. (A. G. A. Coombeset al., Biomaterials 25 (2004) 315-325). Unfortunately, those syntheticscaffolds lack the foam-like consistency, and are of a higher densityand stiffer, and lack several desirable properties, such as porosity tosoak up and retain biological fluids, moldability and compressibility tofit into a hole or defect in bone.

Despite advances in synthetic materials for bone graft applications,there remains a need in the art for further improvements, such asmaterials having tunable properties, lacking animal tissue-derivedmaterials, and which can be manufactured without complex methods.

BRIEF SUMMARY OF THE INVENTION

In accordance with an aspect, a bioactive filamentary structure includesa sheath coated with a mixture of synthetic bone graft particles and apolymer forming a scaffold structure. In some arrangements, thesynthetic bone graft particles may include bioactive (BA) glass and thepolymer may include polycaprolactone (PCL). In some arrangements, alayer of the synthetic bone graft particles may be generally above alayer of the polymer. In some other arrangements, a layer of thesynthetic bone graft particles may be generally below the layer of thepolymer. In some such arrangements, at least some of the synthetic bonegraft particles may extend and be exposed through the layer of thepolymer to promote bone ingrowth upon insertion of the bioactivefilamentary structure into a bone hole at a surgical repair, i.e.,treatment, site.

In some arrangements, the bioactive filamentary structure may define alumen through which a filament, which may be a suture or otherthread-like material, may be passed. In some such arrangements, thebioactive filamentary structure may be constructed of synthetic material(e.g., PLGA, UHMWPE, or the like) or of organic material (silk, animaltendon, or the like).

In accordance with another aspect, a bioactive filamentary structure maybe formed. Synthetic bone graft particles may be applied around afilamentary structure. A polymer solution may be applied around thefilamentary structure. A polymer may be precipitated from the polymersolution such that the synthetic bone graft particles and the polymermay coat the filamentary structure.

In some arrangements, the synthetic bone graft particles may be appliedaround the filamentary structure by placing the filamentary structureinto a container of synthetic bone graft particles and subsequentlyremoving the filamentary structure from the container. In some sucharrangements, the container of the synthetic bone graft particles may beshaken during placement of the filamentary structure into the container.In some such arrangements of applying the synthetic bone graft particlesaround the filamentary structure, the filamentary structure may bedisposed on an inserter, which may be used for later placement of thefilamentary structure, for handling of the filamentary structure duringplacement of the filamentary structure into the container of thesynthetic bone graft particles.

In some arrangements, the polymer solution may be applied around thefilamentary structure prior to applying the synthetic bone graftparticles around the filamentary structure. In some such arrangements,the polymer solution may be applied directly to the filamentarystructure. In some such arrangements of applying the polymer solutionaround the filamentary structure, the polymer solution may be sprayedaround the filamentary structure. In other arrangements, the syntheticbone graft particles may be applied directly to the filamentarystructure prior to applying the polymer solution around the filamentarystructure.

In some arrangements, the synthetic bone graft particles used in formingthe bioactive filamentary structure may include either of or both acalcium phosphate and a bioactive additive. In some arrangements of thesynthetic bone graft particles, the bioactive additive may be but is notlimited to being bioactive glass, bone chips, demineralized bone chipsor powder, living cells, lyophilized bone marrow, collagen, otherbioactive proteins or growth factors, biologics, peptides,glycosaminoglycans, anti-inflammatory compounds, antibiotics,anti-microbial elements, and mixtures of the foregoing. In somearrangements of the synthetic bone graft particles, the calciumphosphate may be but is not limited to being tetra-calcium phosphate,di-calcium phosphate, dicalcium phosphate dihydrous, dicalcium phosphateanhydrous, tri-calcium phosphate, mono-calcium phosphate, β-tricalciumphosphate, a tricalcium phosphate, oxypatite, hydroxypatite, andmixtures of any of the foregoing.

In some arrangements, the polymer precipitated from the polymer solutionused in forming the bioactive filamentary structure may be but is notlimited to being polycaprolactones (PCL), polyglycolides (PGA),polylactic acids (PLA), polyethylene, polypropylene, polystyrene,poly(D,L-lactic-co-glycolide) (PLGA), polyglycolic acid (PGA),poly-L-Lactic acid (PL-LA), polysulfones, polyolefins, polyvinyl alcohol(PVA), polyalkenoics, polyacrylic acids (PAA), polyesters, lower alkylcellulose ethers, methylcellulose, sodium carboxymethyl cellulose,hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethylcellulose, carboxymethyl cellulose, and mixtures ofany of the foregoing.

In some arrangements, the polymer solution used in forming the bioactivefilamentary structure may include at least one solvent. The solvent maybe but is not limited to being glacial acetic acid (GAA), acetic acid,anisole, chloroform, methylene chloride, acetylchloride, 2,2,2trifluoroethanol, trifluoroacetic acid, 1,2-Dochloroethane, and mixturesof any of the foregoing. In preferred arrangements, the polymer solutionmay contain polycaprolactone (PCL) and glacial acetic acid (GAA).

In some arrangements of forming the bioactive filamentary structure, thepolymer may be precipitated from the polymer solution by applying to thepolymer solution a precipitating agent. The precipitating agent may bebut is not limited to being sodium phosphate buffer, water, ethanol,1-propanol, isopropyl ether, 2-butanol, hexane, and mixtures of any ofthe foregoing.

In some arrangements of forming the bioactive filamentary structure, thepolymer may be precipitated from the polymer solution by immersing thepolymer solution in a precipitating agent after applying both thesynthetic bone graft particles and the polymer solution around thefilamentary structure.

In some arrangements of forming the bioactive filamentary structure, thepolymer may be precipitated from the polymer solution by applying afirst buffer to the polymer solution after the polymer solution isapplied around the filamentary structure. In this manner, the polymersolution may be partially neutralized.

In some arrangements of forming the bioactive filamentary structure, asecond buffer may be applied to the polymer solution after the firstbuffer is applied around the filamentary structure to further dilute thepolymer solution. In some such arrangements, the first and the secondbuffers may be sodium phosphate buffers.

In some arrangements of forming the bioactive filamentary structure, thecoated filamentary structure may be dried at least after the applicationof the first buffer to the polymer solution. In some such arrangements,the coated filamentary structure may be dried after the application ofthe second buffer to the polymer solution.

In some arrangements of forming the bioactive filamentary structure, thecoated filamentary structure may be placed into and may be sealed withinsterile packaging, preferably after the coated filamentary structure isdried.

In some arrangements, the coated filamentary structure may be disposedon an inserter used for placing the coated filamentary structure into atreatment site. In some such arrangements, the coated filamentarystructure disposed on the inserter may be placed into and may be sealedwithin sterile packaging, preferably after the coated filamentarystructure is dried.

In accordance with another aspect, a bioactive filamentary structure maybe formed. In this aspect, synthetic bone graft particles may be mixedwith a polymer solution to form a scaffold mixture. The scaffold mixturemay be applied around a filamentary structure. A polymer may beprecipitated from the polymer solution such that the synthetic bonegraft particles and the polymer coat the filamentary structure.

In accordance with another aspect, a bioactive filamentary structure mayinclude a filamentary structure, synthetic bone graft particles, and apolymer. The synthetic bone graft particles may coat the filamentarystructure. The polymer may partially coat the synthetic bone graftparticles such that at least some of the bone graft particles may be atleast partially exposed through the polymer coating. In somearrangements, the filamentary structure may be an all-suture sutureanchor. In some arrangements, the bone graft particles exposed throughthe polymer coating may extending from the filamentary structure beyondthe polymer coating.

Scaffolds and methods of forming the same are disclosed herein. In someembodiments, a method of forming a scaffold includes dissolving apolymer in a solvent to form a polymer solution; adding a precipitatingagent to the polymer solution; precipitating and expanding the polymerfrom the polymer solution to form a scaffold; and removing the solventfrom the scaffold.

In some embodiments, a method of forming a scaffold includes dissolvinga polymer in a solvent to form a polymer solution; adding aprecipitating agent to the polymer solution; precipitating the polymerfrom the polymer solution to form a scaffold comprising an amorphouspolymer matrix; and removing the solvent from the scaffold.

In some embodiments, a method of forming a scaffold includes dissolvinga polymer in a solvent to form a polymer solution; adding aprecipitating agent to the polymer solution; precipitating the polymerfrom the polymer solution to form a scaffold, wherein the time to formthe scaffold by precipitation of the polymer ranges from about 1 minutesto about 1 hour; and removing the solvent from the scaffold.

In some embodiments, a method of forming a scaffold includes dissolvinga polymer in a solvent to form a polymer solution; adding aprecipitating agent to the polymer solution; precipitating the polymerfrom the polymer solution to form a scaffold, wherein a weight ratio ofthe precipitating agent to the polymer solution ranges from about 0.05:1to about 3.5:1; and removing the solvent from the scaffold.

In some embodiments, a scaffold comprises an amorphous polymer matrixhaving a density ranging from about 0.025 g/cm³ to about 0.063 g/cm³.

In some embodiments, a scaffold comprises an amorphous polymer matrixhaving at least one of calcium phosphate or bioactive material embeddedtherein.

In some embodiments, prior to adding the precipitating agent, the methodfurther comprises adding at least one of calcium phosphate or abioactive additive to the polymer solution to form a mixture, whereinafter precipitating the polymer, the scaffold formed includes the atleast one of calcium phosphate or bioactive additive embedded therein.

In some embodiments, an amount of calcium phosphate ranges from about 15weight % to about 40 weight %, based on the total weight of the mixture.

In some embodiments, the method further comprises, prior to adding theat least one of calcium phosphate or bioactive additive to the polymersolution, soaking the at least one of calcium phosphate or bioactivematerial with the precipitating agent to form a precipitatingagent-soaked additive; and freezing the precipitating agent-soakedadditive.

In some embodiments, the method further comprises, prior toprecipitating the polymer, freezing the mixture.

In some embodiments, the precipitating agent is selected from the groupconsisting of water, ethanol, 1-propanol, isopropyl ether, 2-butanol,hexane, and mixtures thereof.

In some embodiments, the method further comprises adding theprecipitating agent drop wise.

In some embodiments, the method further comprises adding theprecipitating agent by spraying or misting the precipitating agent ontoa surface of the polymer solution or the mixture of polymer solution andat least one of calcium phosphate or bioactive additive.

In some embodiments, the method further comprises adding theprecipitating agent by flowing or pouring the precipitation agent ontothe surface of the polymer solution or the mixture of polymer solutionand at least one of calcium phosphate or bioactive additive.

In some embodiments, the polymer solution or the mixture of polymersolution and at least one of calcium phosphate or bioactive additive issubmerged in the precipitating agent.

In some embodiments, the method further comprises, prior toprecipitating the polymer, placing the mixture into a mold.

In some embodiments, the method further comprises, prior toprecipitating the polymer, depositing the mixture on a substrate.

In some embodiments, the substrate is selected from the group consistingof a mesh or screen, a porous polymer substrate, a bone suture anchor, aporous metal implant, living tissue, decellularized tissue, and mixturesthereof.

In some embodiments, prior to precipitating the polymer, the methodfurther comprises soaking the substrate with the precipitating agent.

In some embodiments, the polymer is at least one selected from the groupconsisting of polycaprolactones (PCL), polyglycolides (PGA), polylacticacids (PLA), polyethylene, polypropylene, polystyrene,poly(D,L-lactic-co-glycolide) (PLGA), polyglycolic acid (PGA),poly-L-Lactic acid (PL-LA), polysulfones, polyolefins, polyvinyl alcohol(PVA), polyalkenoics, polyacrylic acids (PAA), polyesters, lower alkylcellulose ethers, methylcellulose, sodium carboxymethyl cellulose,hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethylcellulose, carboxymethyl cellulose, and mixturesthereof.

In some embodiments, the calcium phosphate is at least one selected fromthe group consisting of tetra-calcium phosphate, di-calcium phosphate,dicalcium phosphate dihydrous, dicalcium phosphate anhydrous,tri-calcium phosphate, mono-calcium phosphate, β-tricalcium phosphate,α-tricalcium phosphate, oxypatite, hydroxypatite, and mixtures thereof.

In some embodiments, the calcium phosphate is porous.

In some embodiments, the solvent is at least one selected from the groupconsisting of glacial acetic acid (GAA), acetic acid, anisole,chloroform, methylene chloride, acetylchloride, 2,2,2 trifluoroethanol,trifluoroacetic acid, 1,2-Dochloroethane, and mixtures thereof.

In some embodiments, the bioactive additive is at least one selectedfrom the group consisting of bioactive glass, bone chips, demineralizedbone chips or powder, living cells, lyophilized bone marrow, collagen,other bioactive proteins or growth factors, biologics, peptides,glycosaminoglycans, anti-inflammatory compounds, antibiotics,anti-microbial elements, and mixtures thereof.

In some embodiments, the polymer is polycaprolactone (PCL) and whereinthe solvent is glacial acetic acid (GAA).

In some embodiments, the polymer is polycaprolactone (PCL) and whereinthe solvent is anisole.

In some embodiments, an amount of polymer dissolved in the polymersolution ranges from about 4 wt % to about 15 wt %, relative to thetotal weight of the polymer solution.

In some embodiments, the calcium phosphate is β-tricalcium phosphate(β-TCP).

In some embodiments, an amount of calcium phosphate ranges from about 20wt % to about 30 wt %, relative to the total weight of the mixture.

In some embodiments, the polymer has a molecular weight ranging fromabout 3,000 g/mol to about 150,000 g/mol.

In some embodiments, the time to form the scaffold by precipitation ofthe polymer ranges from about 1 minute to about 1 hour.

In some embodiments, the time to form the scaffold by precipitationranges from about 5 minutes to about 30 minutes.

In some embodiments, a weight ratio of the precipitating agent to thepolymer solution ranges from about 0.05:1 to about 3.5:1.

In some embodiments, a weight ratio of the precipitating agent to thepolymer solution ranges from about 0.5:1 to about 3:1.

In some embodiments, an amount of calcium phosphate ranges from about 15weight % to about 40 weight %, based on the total weight of the mixture.

In some embodiments, removal of the solvent further includes removal ofthe precipitating agent.

In some embodiments, a time for removal of the solvent and/or theprecipitating agent ranges from about 5 minutes to about 3 hours.

In some embodiments, a time for removal of the solvent and/or theprecipitating agent ranges from about 5 minutes to about 1 hour.

In some embodiments, a time for removal of the solvent and/or theprecipitating agent ranges from about 5 minutes to about 30 minutes.

In some embodiments, a time for removal of the solvent and/or theprecipitating agent ranges from about 20 minutes to about 30 minutes.

In some embodiments, the scaffold comprises an amorphous polymer matrix.

In some embodiments, the scaffold comprises at least one of calciumphosphate or a bioactive additive embedded in the amorphous polymermatrix.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription and accompanying drawings in which:

FIG. 1 is a perspective view of a sheath and filament combination of theprior art;

FIG. 2 is a perspective view of a sheath and filament combination inaccordance with an embodiment;

FIG. 3A and 3B are portions of a process flow diagram for preparing thesheath and filament combination shown in FIG. 2;

FIGS. 4A and 4B are perspective views of the sheath and filamentcombination shown in FIG. 2 in pre-deployment and deployed states;

FIGS. 5A and 5B are plan views of respective portions of sheaths exposedto simulated body fluid in accordance with other embodiments;

FIG. 6 is a perspective view of a sheath and filament combination inaccordance with another embodiment; and

FIG. 7 is a perspective view of a portion of a process flow diagram forpreparing the sheath and filament combination shown in FIG. 6.

FIGS. 8A-8D depict scaffolds made using a range of concentrations for apolymer solution in accordance with some embodiments of the presentinvention.

FIGS. 9A-9F depict scaffolds made using a range of amounts of a polymersolution in accordance with some embodiments of the present invention.

FIG. 10 depicts a scaffold in accordance with one embodiment of thepresent invention.

FIG. 11 depicts a scaffold in accordance with another embodiment of thepresent invention.

FIGS. 12A-12D depict scaffolds in accordance with some embodiments ofthe present invention.

FIG. 13A depicts a substrate in accordance with some embodiments of thepresent invention.

FIG. 13B depicts a scaffold including the substrate of FIG. 13A inaccordance with some embodiments of the present invention.

FIG. 14A depicts a substrate in accordance with some embodiments of thepresent invention.

FIG. 14B depicts a scaffold including the substrate of FIG. 14A inaccordance with some embodiments of the present invention.

FIG. 14C depicts a scaffold including the substrate of FIG. 14A inaccordance with some embodiments of the present invention.

FIG. 14D depicts a scaffold including the substrate of FIG. 14A inaccordance with some embodiments of the present invention.

FIG. 15 depicts high magnification image of the scaffold depicted inFIG. 14B.

FIG. 16A depicts a scanning electron microscopy (SEM) image of thescaffold of FIG. 14C.

FIG. 16B depicts an EDAX (energy dispersive x-ray) chemical map of theSEM image of FIG. 16A.

FIG. 16C depicts EDAX data of the scaffold of FIG. 14C.

FIGS. 17A-17B depict SEM images of a scaffold in accordance with someembodiments of the present invention.

FIG. 18 depicts EDAX data from a region of the scaffold depicted in FIG.17A.

FIG. 19 depicts an SEM image of a scaffold in accordance with someembodiments of the present invention.

FIG. 20A depicts EDAX data from regions of a scaffold in accordance withsome embodiments of the present invention.

FIG. 20B depicts EDAX data from regions of the scaffold depicted in FIG.19 in accordance with some embodiments of the present invention.

FIG. 21A depicts scaffolds formed using different amounts ofprecipitating agent in accordance with some embodiments of the presentinvention.

FIG. 21B depicts the thickness variation of the scaffolds depicted inFIG. 21A as function of the amount of precipitating agent added inaccordance with some embodiments of the present invention.

FIG. 22 depicts scaffolds formed using different orders of processingsteps in accordance with some embodiments of the present invention.

DETAILED DESCRIPTION

As used herein, the term “filament” and like terms are inclusive ofsingle or multiple strands, threads, fibers, strings, wires or suturesin which such terms preferably refer to a suture or other thread-likematerial, and in particular a braided suture, having a hollow core alongat least a portion of its length. A filament may be constructed fromhomogenous or heterogeneous materials such as, but not limited to,polyester, polyethylene (including ultra-high molecular weightpolyethylene (UHMWPE)), polytetrafluorethylene (including expandedpolytetrafluorethylene), nylon, polypropylene, aramids (such asKevlar-based materials), polydioxanone, polygycolic acid, liquid crystalpolymer (LCP), organic material (silk, animal tendon, or the like),metallic wire, or any combination of these materials.

Referring now to the drawings, as shown in FIG. 1, sheath and filamentcombination 10 known in the art forms an all-suture suture anchor thatincludes first filament 15A and second filament 15B, which in somearrangements may be a monofilament while in other arrangements may be abraided suture as shown, and sheath 20 having an inner lumen defined byinner surface 27 of the sheath through which the filament is inserted.Preferably, each of sheath 20 and the filaments 15A, 15B are composed ofsuture, and specifically a blend of UHMWPE and polyester. Sheath 20,which may be but is not limited to being a sheath of an all-suturesuture anchor for use as part of the ICONIX® All Suture Anchor System,includes openings 25 along its length that expose filaments 15A, 15B andallow the sheath to fold upon itself and to compress when opposingportions of the filament are pulled in a direction away from the sheathwhile the sheath is held, such as by a patient's bone or other tissue,at its ends. This deformation of the sheath drastically changes anaspect ratio of the sheath to provide a resistance to pullout when thesuture is inserted into a prepared bone hole (see FIG. 4B for an exampleof this deformation being applied to prepared sheath 120 prepared inaccordance with an embodiment of the present invention), as described inU.S. Pat. No. 9,445,803 to Marchand et al. (“the '803 Patent”), which ishereby incorporated by reference in its entirety herein.

As shown in FIG. 2, sheath and filament combination 110 includes firstfilament 15A, second filament 15B, and prepared sheath 120 having aninner lumen through which the filament is inserted. Prepared sheath 120is generally formed by applying synthetic bone graft particles, whichmay be but are not limited to being bioactive (BA) glass or calciumphosphate particles, onto and within sheath 20 and then coating thesynthetic bone graft particles with a polymer coating layer, which maybe but is not limited to being a layer of polycaprolactone (PCL). ThePCL acts as a barrier or otherwise secures the synthetic bone graftparticles to prevent migration and rapid degradation and diffusion ofthe bone graft particles upon insertion of sheath and filamentcombination 110 into a treatment site.

Referring now to FIGS. 3A and 3B, in step 130, polymer solution 135containing the polymer coating and a solvent in which the polymercoating may be soluble, which preferably may be acetic acid (AA) andmore preferably may be glacial acetic acid (GAA), may be prepared bymixing the polymer coating in the solvent at an appropriate temperaturefor an appropriate time. In preferred arrangements, the polymer may befully dissolved in the solvent. In one example, to form polymer solution135, the mixture of the polymer and the solvent preferably may be placedon a heated plate, such as but not limited to the plate of an orbitalshaker, set at a temperature in the range of approximately 30° C.to—approximately 55° C. for a time preferably less than 24 hours, orotherwise up until a time the polymer fully dissolves.

Once prepared, the molecular weight (MW) of the polymer coating may bebut is not limited to being in the range of 33-117 kilodaltons (kDa). Ingeneral, greater MWs of the polymer coating will lead to greaterbioactivity at a treatment site but will cause slower resorbability.However, bioactivity may vary with other variables such as but notlimited to any of the amount, size, and surface area of the syntheticbone graft particles, coating thickness and porosity. In preferredarrangements, the concentration of polymer solution 135 may be in therange of about 5% to about 16% mass per unit volume, and preferablyapproximately 5%. The viscosity of polymer solution 135 may be desirablyin the range of approximately 1 to approximately 30 centipoise (cP), andpreferably about 20 cP. Within this viscosity range, polymer solution135 may have sufficient flowability for application to sheath 20 whileat the same time have sufficient adhesion characteristics to inhibitsliding of the solution off of the sheath after application. Alterationsin concentration and molecular weight of polymer solution 135 may havean effect on droplet size, spray pattern, and the characteristics of thepolymer coating after being applied to sheath 20.

In parallel with the preparation of polymer solution 135, sheath 20 iscoated with synthetic bone graft particles, which may be but are notlimited to being BA glass particles, at step 140. In some arrangements,sheath 20 may be held horizontally (i.e., parallel to the floor), suchas by an operator or a robotic arm, and dipped into a containercontaining the bone graft particles. In such arrangements, openings 25of sheath 20 may be oriented vertically upward (i.e., face away from thefloor) during dipping of the sheath into the container such that thesynthetic bone graft particles contact and enter only or at least mainlyouter surface 26 (as illustrated in FIG. 1) of the sheath. Inalternative arrangements, sheath 20 may be held in any otherorientation, including vertically in which when the sheath is dippedinto the container of the synthetic bone graft particles, such particlesmay coat at least a portion of inner surface 27 (identified in FIG. 1)of the sheath and may be held on the anchor by ionic forces, van derWaals' forces and physical entrapment between bone graft particles. Suchinteractions may be generated by static electricity caused by frictionbetween the bone graft particles and the sheath during the dipping ofthe sheath. In some arrangements, the bone graft particles may be heldmechanically by being placed in the interstices of the fibers of thesheath. In some alternative arrangements, a resistive mask (not shown),which may be but is not limited to being a tape, film, or othercovering, may be applied over central openings 25 and end openings 29 ofsheath 20 to prevent or at least inhibit the intrusion of synthetic bonegraft particles into the inner lumen of the sheath defined by innersurface 27 of the sheath. In some arrangements, the resistive mask mayprevent friction between first filament 15A and second filament 15B.

In preferred arrangements, sheath 20 may be dipped into the syntheticbone graft particles while surrounding filaments 15A, 15B as part ofsheath and filament combination 10, while in other arrangements, sheath20 may be separated from either or both of filaments 15A, 15B during thegraft coating process. In arrangements in which sheath 20 is orientedwith either of or both central openings 25 and end openings 29 exposedto the synthetic bone graft particles, at least a portion of either offilaments 15A, 15B may be coated with the synthetic bone graft particlesalong with sheath 20. To avoid coating either or both of filaments 15A,15B or allowing synthetic bone graft particles to be incorporatedbetween either of the filaments and the interior of sheath 20, aresistive mask (not shown) may be applied to the sheath over openings 25during application of the synthetic bone graft particles onto thesheath. Reducing these particles in these areas may avoid introducingadditional friction when sliding filaments 15A, 15B through sheath 20after insertion of sheath and filament combination 10 into a preparedbone hole at a treatment site.

As shown in FIG. 3A, at step 139 prior to step 140, sheath and filamentcombination 10 may be placed onto an inserter (not shown), such as theinserter described in the '803 Patent or the inserter described ineither of U.S. Pat. No. 8,821,494 B2 to Pilgeram and U.S. PatentApplication Publication No. 2014/0039552 A1 to Pilgeram, the disclosuresof each of which are hereby incorporated by reference herein. In thismanner, the inserter may be held by the respective operator or roboticarm during placement of sheath 20, or in some arrangements sheath andfilament combination 10, into the container of synthetic bone graftparticles. Placing sheath 20 onto the inserter prior to loading withsynthetic bone graft particles avoids any loss of the particles that mayotherwise occur during such placement of the sheath. The particle sizeof the synthetic bone graft particles applied to sheath 20 is preferablyin the range from about 32 μm to about 90 μm. Larger or smallerparticles sizes are possible in which smaller particle sizes drive morebioactivity at a treatment site but may be resorbed too quickly forcertain applications.

In some arrangements, the container of synthetic bone graft particlesmay be in the form of a bowl or cup, such as but not limited to a glass,plastic, or metal bowl. In some arrangements, either of or both thecontainer and sheath 20 (and in some instances, the inserter andfilament 15 as described previously herein) may be vibrated to preventor inhibit agglomeration of the particles and to achieve an appropriategraft coating mass for a particular anchor size. In some arrangements,the container may be rotated, i.e., spun, and may be translated alongwith being vibrated. Vibration, translation, and rotation of thecontainer allows the synthetic bone graft particles to uniformly,thoroughly, and continuously contact the outer surface of sheath 20. Inthis manner, the bone graft particles may coat sheath 20 and becomewedged between fibers forming the sheath. The frequency and amplitude ofeither of or both the vibration and rotation may be altered by changinga corresponding voltage setting on a vibration fixture, which may be butis not limited to being a vibration table. These settings along with thetime of vibration affect the mass of the synthetic bone graft particlesapplied to sheath 20. In this manner, the synthetic bone graft particlesmay be embedded between the fibers of sheath 20, and preferablyagglomerates at pics of the fibers of the anchor. Sheath 20 may betensioned and compressed along an axis, e.g., its longitudinal axis, toopen the fibers of the sheath. In this manner, the synthetic bone graftparticles may be set in between the fibers. Alternatively, sheath 20 mayany of be vibrated, translated, and rotated relative to the container inorder to coat the sheath with the synthetic bone graft particles. Insome arrangements, after graft particles are applied to sheath 20, thesheath (along with filament 15 and the inserter if attached to thesheath) may be shaken to remove loose particles.

With reference to step 150 shown in FIG. 3A, graft-coated sheath 20 maybe coated with polymer solution 135. Polymer solution 135 may coat, inone example, at least 30% of the outer surface of graft-coated sheath20, in another example, at least 70% of the outer surface ofgraft-coated sheath, and, in still another example, at least 90% of theouter surface of graft-coated sheath, although in some arrangements, thepolymer solution may coat less than 30% of the outer surface of thegraft-coated sheath. In the example shown, polymer solution 135 may besprayed, for example in the form of a mist, from a nozzle (not shown)onto some, or preferably all exposed areas, of graft-coated sheath 20.In some arrangements, the sprayer may be mechanical sprayer, in whichthe flow rate provided by the sprayer may be altered by increasing ordecreasing the restriction within the nozzle of the sprayer. In otherarrangements, the sprayer may be part of an ultrasonic spray system,e.g., the Sono-tek ExactaCoat SC ultrasonic spraying system, whichincludes an ultrasonic spray nozzle used in conjunction with an airstream directed to the nozzle and controlled with a gantry system, whichmay be operated by server motors, or other motion control system. Insuch arrangements, the flow rate of the spray as it exits the nozzle maybe altered as desired by adjusting the voltage to piezoelectrictransducers to create vibrations against and to cause the atomization ofpolymer solution 135 as it flows.

The flow rate of the spray, the local air pressure around the spray, thedistance of the exit of the nozzle from graft-coated sheath 20 (oruncoated sheath 20 in other embodiments such as in the formation ofprepared sheath 220 described further herein), the speed of the exit ofthe nozzle itself relative to the sheath, and the number of passes overan area or areas of the sheath all affect the thickness of polymersolution 135 applied to the graft-coated (or uncoated) sheath.Preferably, polymer solution 135 should not be too thin when applied tograft-coated (or uncoated) sheath 20 such that the solution does notsufficiently adhere to the respective coated (or uncoated) sheath.Conversely, polymer solution 135 should not be too thick such thateither of or both sheath and filament combination 110 is too stiff andprepared sheath 120 (or other prepared sheath, such as prepared sheath220) defines a maximum outer perimeter that is greater than an innerperimeter of a prepared bone hole into which the sheath is to beinserted and thus is susceptible to removal of the applied syntheticbone graft particles during such insertion.

Referring now to FIG. 3B, during step 160, polymer solution 135 coatingsheath 20 is exposed to a precipitating agent, which may be but is notlimited to being a sodium phosphate buffer composed of sodium monobasicand sodium dibasic in water (Na₂HPO_(4 and) NaH₂PO₄) or other neutral pHsolution, which causes precipitation of the polymer coating from polymersolution 135 such that the polymer coating may coat the bone graftparticles of prepared sheath 20 in order to retain the particles andprevent their premature degradation when prepared sheath 120 is insertedinto a treatment site. In preferred arrangements, sheath 20 coated withsolution 135, which in more preferred arrangements may be disposed on aninserter as discussed previously herein, is dipped into a reservoircontaining the precipitating agent. In some alternative arrangements,the precipitating agent may be sprayed onto sheath 20 coated withpolymer solution 135. Under certain of the conditions of the nozzlelisted above with respect to the thickness of polymer solution 135, astream, i.e., a fluid jet, of 10 mL to 20 mL of the buffer solution maybe rapidly applied for less than approximately 30 seconds to sheath 20prepared with polymer solution 135, again which in more preferredarrangements may be disposed on an inserter as discussed previouslyherein. In any of these arrangements, sheath 20 when coated with polymersolution 135 preferably is soaked or otherwise saturated with theprecipitating agent immediately after application of polymer solution135 in order to more evenly distribute the polymer solution and toreduce the exposure of the polymer solution, the synthetic bone graftparticles when such particles are coating sheath 20, and the sheathitself to acidic conditions.

During step 170 and following precipitation step 160, sheath 20 coatedwith the polymer coating and remaining polymer solution 135 is washed,such as by but not limited to being by either of or both a sodiumphosphate buffer and deionized (DI) water (“wash solution”) . In thismanner, residual salts that have formed, which may be acetate andphosphate salts from the GAA (or other form of AA) solvent used inpolymer solution 135 and the sodium phosphate buffer used at step 160,are reduced to a physiologically acceptable range. In preferredarrangements, sheath 20 coated with the polymer coating and remainingpolymer solution is washed in a bath containing the wash solution. Thecombination of precipitation step 160 and washing step 170 under properconditions should yield a residual amount of the GAA (or other form ofAA) solvent of preferably less than approximately 0.040 molar (M). Useof the buffer as or in the washing solution neutralizes the pH of thecoating on sheath 20 more quickly during processing than water alone andthus prevents unnecessary erosion of the synthetic bone graft particlesotherwise caused by the GAA (or other form of AA) solvent.

As further shown in FIG. 3B, during step 180, sheath 20 coated with theprecipitated polymer coating covering the applied synthetic bone graftparticles is subjected to drying to form prepared sheath 120 and toremove any residual GAA (or other form of AA). In this manner,degradation of the synthetic bone graft particles and polymer coatingdue to the GAA (or other form of AA) during packaging may be preventedto extend the shelf life of sheath 120. Preferably, drying step 180 maybe conducted without the addition of heat when used with polymers havinglow melting points, such as but not limited to PCL having a meltingpoint of approximately 60° C. In some preferred arrangements, coatedsheath 20 may be dried by vacuum to quickly remove any moisture andresidual acid while preserving the integrity of the synthetic bone graftparticles and resultant polymer coating. It is also possible to drycoated sheath 20 with air at atmospheric pressure or forced air at anelevated pressure. Supplying compressed or otherwise forced air tocoated sheath 20 is also less preferable than applying a vacuum as asufficient amount of air to remove an acceptable amount of moisture maycause some of the polymer coating or synthetic bone graft particles tobe removed from sheath 20. Following drying step 180, the moisturecontent within the polymer coating is preferably less than approximately0.5% wt, and the synthetic bone graft particles are firmly adhered tosheath 20 by the remaining polymer coating. Optionally, prior to or, insome arrangements, in place of drying step 180, sheath 20 may be driedby way of moisture wicking through a capillary effect via direct contactwith moisture on the sheath using a porous-like structure, such as butnot limited to a brush, a cloth or paper towel, by way of a desiccant,or by way of another acceptable and preferably biocompatible dryingagent known to those of ordinary skill in the art .

In preferred arrangements, after precipitation and drying of the polymercoating, the thickness of the polymer coating of prepared sheath 120 (orother prepared sheath such as prepared sheath 220) preferably may be inthe range of less than approximately 100 μm, and more preferably in therange of 1 μm to 30 μm. As such, the polymer coating may have athickness such that a significant number of synthetic bone graftparticles coating sheath 20 are partially exposed, at their apices,through the polymer coating in addition to their exposure through thepores of the polymer coating. By way of this exposure, bioactivity mayoccur shortly after placement of prepared sheath 120 at a treatmentsite.

Referring now to FIG. 4A, when initially prepared as sheath and filamentcombination 110 placed onto the inserter, prepared sheath 120 is foldedonto itself such that opposing portions 121, 122 of the prepared sheath,which may be in the form of legs, extend from end 123 in substantiallyparallel directions to each other. As shown in FIG. 4B, when deployed,prepared sheath 120 compresses, i.e., bunches, such that opposingportions 121, 122 are pushed in transverse directions to the inserterand away from each other, forming a substantially clover-like shape. Inthis deployed, compressed state, portions of the polymer coating may anyof break up, crack, and stretch, providing further exposure of thesynthetic bone graft particles and thus providing for greaterbioavailability.

As further shown in FIG. 3B, during step 190, prepared sheath 120 isinserted into sterile packaging. In some arrangements, prepared sheath120 may be placed into a pouch, which may be but is not limited to beingmade of aluminum foil, with a covering or leader, which may be but isnot limited to being an olefin sheet such as but not limited to Tyvek®olefin sheets by E. I. du Pont de Nemours and Company. In somearrangements, the sheath (or sheath and filament combination) inserterdescribed previously herein may be placed into the pouch with preparedsheath 120 disposed on the inserter and ready for placement into atreatment site. In alternative arrangements, prepared sheath 120 (orsheath and filament combination 110) may be separated from the inserter,and in such arrangements, the inserter may be placed in the same pouchas the sheath (or respective sheath and filament combination 110) or aseparate sterile pouch. In any of these arrangements, a plurality ofcombinations or kits of prepared sheath 120 (or respective sheath andfilament combination 110) and the inserter may be placed into sleeves.Preferably, when packaged and sterilized, the sterility assurance level(SAL) for prepared sheath 120 (or respective sheath and filamentcombination 110), or for the combination or kit of the sheath (orrespective sheath and filament combination 110) and the correspondinginserter, is at or below 10⁻⁶ SAL To achieve this SAL, the packagedsheath 120 or packaged combination or kit of the sheath and thecorresponding inserter preferably may be subjected to ethylene oxide(EtO) processing as well as periodic functional checks and lot releasetesting.

As shown in FIG. 5A, in one arrangement, sheath and filament combination110A may be as described previously herein with respect to sheath andfilament combination 110 in which the polymer coating is low molecularweight (LMW) PCL. As shown, the LMW PCL leads to hydroxyapatitedeposition and crystallization (shown in whitish gray) on the surface ofthe sheath when exposed to living tissue fluids (as demonstrated throughthe use of simulated body fluids in the example shown), in particular atthe pics, i.e., crossings, of fibers of the sheath. As shown in FIG. 5B,in another arrangement, sheath and filament combination 110B asdescribed previously herein with respect to sheath and filamentcombination 110 in which the polymer coating is high molecular weight(HMW) PCL. The HMW PCL leads to hydroxyapatite deposition andcrystallization (shown in whitish gray) on the surface of the sheathwhen exposed to living tissue fluids (as demonstrated through the use ofsimulated body fluids in the example shown), like the LMW PCL, but thehydroxyapatite deposition is substantially greater for the HMW PCL thanfor the LMW PCL, as shown by comparison of FIGS. 5A and 5B.

Referring now to FIG. 6, in another arrangement, sheath and filamentcombination 210 includes first filament 15A, second filament 15B, andprepared sheath 220 having an inner lumen through which the filamentsare inserted. Prepared sheath 220 is generally formed by coating sheath20 (see FIG. 1) with a polymer coating layer and then applying syntheticbone graft particles onto the polymer-coated sheath 20. In this manner,it is possible to add more exposed synthetic bone graft particles tosheath 20.

Referring now to FIG. 7, prepared sheath 220 is formed in the same orsubstantially the same manner as prepared sheath 120 with twoexceptions. First, polymer solution coating step 150 is replaced withpolymer solution coating step 250 in which polymer solution 135 coatssheath 20 without any synthetic bone graft particles coating the sheath.Second, graft coating step 140 is replaced with graft coating step 240in which synthetic bone graft particles are applied to sheath 20 coatedwith polymer solution 135.

In some alternative arrangements, in place of or in addition to BAglass, the synthetic bone graft particles may include but are notlimited to calcium phosphate or other bioactive additives. The calciumphosphate may be but is not limited to being tetra-calcium phosphate,di-calcium phosphate, dicalcium phosphate dihydrous, dicalcium phosphateanhydrous, tri-calcium phosphate, mono-calcium phosphate, β-tricalciumphosphate, a-tricalcium phosphate, oxypatite, hydroxypatite, andmixtures thereof. The other bioactive additives may include but are notlimited to bone chips, demineralized bone chips or powder, living cells,lyophilized bone marrow, collagen, other bioactive proteins or growthfactors, biologics, peptides, glycosaminoglycans, anti-inflammatorycompounds, antibiotics, anti-microbial elements, and mixtures thereof.

In some alternative arrangements of sheath and filament combination 110,in place of or in addition to PCL, the barrier layer may be but is notlimited to being replaced with another polymer such as polyglycolides(PGA), polylactic acids (PLA), polyethylene, polypropylene, polystyrene,poly(D,L-lactic-co-glycolide) (PLGA), polyglycolic acid (PGA),poly-L-Lactic acid (PL-LA), polysulfones, polyolefins, polyvinyl alcohol(PVA), polyalkenoics, polyacrylic acids (PAA), polyesters, lower alkylcellulose ethers, methylcellulose, sodium carboxymethyl cellulose,hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethylcellulose, carboxymethyl cellulose, and mixturesthereof.

In some alternative arrangements, in place of or in addition to GAA, thecoating solvent in the PCL or other polymer solution may be but is notlimited to being at least one solvent of any of acetone, anisole,chloroform, methylene chloride, acetylchloride, 2,2,2 trifluoroethanol,trifluoroacetic acid, 1,2-Dochloroethane, mixtures thereof.

In some alternative arrangements, precipitating agents other than or inaddition to the sodium phosphate buffer described previously herein maybe used during formation of coated sheaths such as sheaths 120, 220.Such precipitating agents include but are not limited to water, ethanol,1-propanol, isopropyl ether, 2-butanol, hexane, and mixtures thereof.

In some alternative arrangements, the combination of the polymer coatingand the synthetic bone graft particles may be applied to any braidedstructure, especially such structures to be implanted into bone. Suchbraided structures may be but are not limited to ligament graftmaterial, e.g., anterior cruciate ligament (ACL) graft material. Thecombination of the polymer coating and the synthetic bone graftparticles may be applied to any such braided structure in the samemanner that the combination of the polymer coating and the syntheticbone graft particles described previously herein as being applied tosheath and filament combination 110, 210.

All percentages and ratios used hereunder are by weight of the totalcomposition and all measurements made are at about room temperature andnormal pressure unless otherwise designated. “Room temperature” asdefined hereunder means a temperature ranging between about 22° C. andabout 26° C. All temperatures hereunder are in degrees Celsius unlessspecified otherwise.

As used herein, “consisting essentially of” means that the invention mayinclude ingredients in addition to those recited in the claim, but onlyif the additional ingredients do not materially alter the basic andnovel characteristics of the claimed invention. Preferably, suchadditional ingredients will not be present at all or only in traceamounts. However, it may be possible to include up to about 10% byweight of materials that could materially alter the basic and novelcharacteristics of the invention as long as the utility of the compounds(as opposed to the degree of utility) is maintained.

All ranges recited herein may include the endpoints, including thosethat recite a range “between” two values. Terms such as “about,”“generally,” “substantially,” and the like are to be construed asmodifying a term or value such that it is not an absolute, but does notread on the prior art. Such terms will be defined by the circumstancesand the terms that they modify as those terms are understood by those ofskill in the art. This includes, at very least, the degree of expectedexperimental error, technique error and instrument error for a giventechnique used to measure a value.

It should be further understood that a description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of the invention. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible sub-ranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 2.3, 3, 4, 5, 5.7 and 6. This appliesregardless of the breadth of the range.

The terms such as “at least one of calcium phosphate or bioactiveadditive”, “calcium phosphate and/or bioactive additive”, and “bioactiveadditive and/or calcium phosphate” are used interchangeably throughoutthe remainder of the detailed description of this specification andshould be understood to mean “calcium phosphate without bioactiveadditive, bioactive additive without calcium phosphate, or both calciumphosphate and bioactive additive.”

The terms “weight percent of the polymer solution” or “polymerconcentration” and “weight percent of the mixture” as used herein havedifferent meanings. The terms “weight percent of the polymer solution”or “polymer concentration” are defined as the weight of the polymerrelative to the weight of the solvent. For example, a 10 wt % PCL-GAAsolution may have 10 grams of PCL relative to 100 grams of GAA. Incontrast, the term “weight percent of the mixture” is defined as theweight of a component of the mixture relative to the total weight of themixture including the component. For example, a mixture of calciumphosphate and 10 wt % PCL-GAA solution having an amount of 10 wt %calcium phosphate may be 10 grams of calcium phosphate to 100 grams ofthe total weight of the mixture, i.e., the summed weight of calciumphosphate and 10 wt % PCL-GAA solution. The term “weight ratio” as usedherein is defined as the weight of one component to the weight ofanother component. For example, a weight ratio of 6 grams of water to 2grams of a 10% PCL-GAA solutions is 6:2.

The term ‘molecular weight’ as used herein in regards to a molecularweight of a polymer is a weight average molecular weight (M_(w)) unlessotherwise specified. If a number average molecular weight of a polymeris used, it is indicated as M_(n).

The terms ‘foam’, loam-like', ‘foaming’ as used herein are not intendedto describe a scaffold comprising a classically defined foam or formedby foaming, where a classically defined foam comprises a mass of smallbubbles in a liquid. The terms ‘foam’, foam , or ‘foaming’ as usedherein describe a physical appearance of a scaffold as similar to thatof Styrofoam or seam-foam.

The terms “expansion”, or “expanding” as used herein to describe anincrease in a volume or space that a group of polymer molecules occupyupon or while precipitating from a polymer solution or an increase inthe volume of the scaffold formed upon precipitation. A scaffold formedfrom a precipitated and expanded polymer initially present in a polymersolution, or a precipitated and expanded polymer initially present in amixture including the polymer solution and at least one of calciumphosphate or a bioactive additive, can have a structure that isfoam-like and/or amorphous.

METHODS

Described herein are methods of making a scaffold in accordance withsome embodiments of the present invention. In one embodiment, a methodof preparing a scaffold may include dissolving a polymer in a solvent toform a polymer solution; adding a precipitating agent to the polymersolution; precipitating and expanding the polymer from the polymersolution to form the scaffold; and removing the solvent from thescaffold.

The method described herein can employ a two phase system to form ascaffold. The two phase system may comprise a polymer solution as onephase and precipitating agent as the other phase. Chemical reactivityresulting from the precipitating agent being introduced into the polymersolution induces rapid intermixing, precipitation and expansion of thepolymer to form the scaffold. Scaffolds can be formed within minutes andcan be relatively homogeneous, amorphous, non-crystalline, foam-like,and porous, having irregularly-sized holes and channels. The method doesnot use a foaming agent to achieve a scaffold having a foam-likestructure. In one embodiment, the polymer solution can be a viscoussolution, such as PCL dissolved in glacial acetic acid. In oneembodiment, the precipitating agent can be water, a non-organic lowviscosity solvent.

The polymer may include one or more polymers as discussed herein. Asolvent may include any suitable solvent that is capable of dissolvingthe polymer, and may vary depending on the polymer that is beingdissolved. Exemplary solvents may include, without limitation, one ormore of glacial acetic acid (GAA), anisole, chloroform, methylenechloride, acetylchloride, 2,2,2 trifluoroethanol, trifluoroacetic acid,and/or 1,2-Dochloroethane. Other solvents that may be utilized with theinventive methods may be found in Bordes, C., et al., 2010,International J. Pharmaceutics, 383, 236-243, which is incorporatedherein by reference.

The polymer can be dissolved in the solvent in any suitableconcentration necessary to produce a scaffold having the desiredproperties. The concentration of polymer in the polymer solution mayinfluence mechanical properties of the resulting scaffold, such asflexibility and ability to retain calcium phosphate or bioactiveadditive. In some embodiments, physical and chemical properties of thepolymer solution, such as molecular weight, specific gravity, polarity,or the like, may have an influence on the morphology of the scaffoldformed therefrom. In some embodiments, morphology of the scaffold may bemodified, at least partially, by identity of polymer and/or solvent.

The concentration of polymer in solution may depend on the polymer andsolvent used. For example, the solubility of PCL in many differentsolvents was reported in Bordes, C., et al., where it is noted thatdepending on the solvent used, PCL having a range of molecular weightsmay be soluble at up to 50 wt % solutions. In some embodiments, thepolymer concentration may range from about 1 wt % to about 50 wt %,relative to the weight of solvent, preferably from about 5.0 wt % toabout 15 wt %, more preferably from about 7.0 wt % to about 10 wt % ofthe polymer solution. The polymer concentration range may be above 50 wt% in some embodiments. In some embodiments, the polymer may be presentin a saturated amount or supersaturated amount in the polymer solution.In some embodiments, the polymer may be PCL and the solvent may be GAA,where PCL is dissolved in GAA ranging from about 7.0 wt % to greaterthan about 10 wt %, or to a saturated level. In some embodiments, thesaturated level may be in excess of about 10 wt % but less that about 15wt % for PCL dissolved in GAA. As shown in FIGS. 8A-8D, PCL solutionsranging from about 7.0 wt % to up to a saturated level (labeled as10%-15%) can produce scaffolds that retain a shape of a mold in whichthey were made, and retain calcium phosphate, which is embedded in thescaffold during the molding process. In some embodiments, higherconcentrations of PCL in solution may be achieved using anisole as asolvent, instead of GAA. In such an embodiment, PCL may be dissolved inanisole from about 10 wt % to about 50 wt %. For example, PCL having amolecular weight of about 14,000 g/mol was soluble in anisole at up to50% wt %, and PCL having a molecular weight of 65,000 g/mol may bedissolved at 10 wt % but less than 50wt %.

The method may include precipitating the polymer from the polymersolution to form the scaffold. Alternatively, when calcium phosphateand/or bioactive additive is used (described further below), the polymermay be precipitated from a mixture formed from calcium phosphate and/orbioactive additive added to the polymer solution. The precipitation ofthe polymer from the polymer solution may generate a scaffold that isfluffy, amorphous, absorbent, and low density, unlike higher densityscaffolds generated using other methods. A low density scaffold may bemore porous, deformable, drapable, compressible, not load bearing to asignificant extent, and able to tear apart by hand, in contrast to ahigh density scaffold. A high density scaffold would be less porous,somewhat load bearing, rigid and marginally, if at all drapable, notvery compressible, and difficult, if not impossible to tear apart byhand, compared with a low density scaffold. In some embodiments,scaffolds of medium or high density can also be achieved using methodsdiscussed herein. In some embodiments, the scaffolds can resist handpressure to some extent as well as retaining fluid even when a force tenor more times its weight is applied. In some embodiments, compressionresistance of the scaffold may depend on any or all of polymerconcentration in the polymer solution, polymer molecular weight, andamount of calcium phosphate and/or bioactive additive used.

The polymer may be precipitated by contacting the polymer solution orthe mixture with a precipitating agent to form the scaffold. In someembodiments, the entire scaffold can be formed by contacting the polymersolution or mixture with the precipitating agent. For example, thescaffold is not formed piecewise by using a precipitating agent toprecipitate a portion of the polymer, and removing solvent toprecipitate another portion of the polymer. In some embodiments, ascaffold having a foam-like and/or amorphous structure is not formed inthe absence of a precipitating agent. For example, removal of solvent toprecipitate the polymer in the absence of a precipitating agent does notform a scaffold having a foam-like and/or amorphous structure. Exemplaryprecipitating agents can include, without limitation, water, ethanol,1-propanol, isopropyl ether, 2-butanol, hexane, or combinations thereof.The total time to precipitate a scaffold can be on the order of minutes.In some embodiments, a precipitation time may range from about 1 minuteto about 30 minutes. In other embodiments, the precipitation time mayrange from about 5 minutes to 25 minutes. In yet other embodiments, theprecipitation time may range from 10 minutes to 20 minutes. In yet otherembodiments, a precipitation time up to 1 hour may be utilized.

The polymer may be precipitated in various ways. For example, in someembodiments where a mold is used, the precipitating agent may beinitially added drop wise to an exposed surface of the polymer solutiondisposed in the mold. The mold may, optionally, then be submerged in theprecipitating agent. In some embodiments, when the polymer solution ispresent on a substrate, the polymer solution including the substrate maybe immersed in the precipitating agent to precipitate the polymer toform the scaffold. In some embodiments, the precipitating agent may besprayed or misted onto a surface of the polymer solution or a mixtureincluding the polymer solution and at least one of calcium phosphate orbioactive additive. In some embodiments, the precipitating agent may beflowed or poured onto the surface of the polymer solution or the mixtureof polymer solution and at least one of calcium phosphate or bioactiveadditive. The amount of precipitation agent added and/or its speed ofaddition may strongly influence the foam-like qualities and density ofthe resulting scaffold.

In some embodiments, an order in which method steps are performed maydetermine the morphology of a resulting scaffold. One exemplaryembodiment is described in Experimental Example 6 below. Under someconditions, a scaffold formed by dropwise adding of a precipitatingagent to a polymer solution may result in a fluffy, amorphous, foam-likescaffold. Under some conditions, when the same amount of polymersolution is dropwise added to a precipitating agent, the resultingscaffold can be irregularly shaped and has non-homogenous density.

The polymer can expand upon or while precipitating from the polymersolution or a mixture including the polymer solution and at least one ofcalcium phosphate or a bioactive active. In some embodiments,precipitation and expansion of the polymer occur simultaneously. In someembodiments, the amount of precipitating agent and/or the speed at whichit is added may cause both precipitation and expansion of the scaffold.One exemplary embodiment is described in Experimental Example 6. When noprecipitating agent is used, and the scaffold is precipitated throughair drying, the scaffold is dense, slightly flexible when manipulated byhand, and appears to have little or no porosity based on visualinspection. As an amount of precipitating agent is increased relative tothe amount of PCL solution, the resulting scaffold is less dense,porous, fluffy, and Styrofoam-like in appearance. If the amount ofprecipitating agent is increased too far relative to the amount of PCLsolution, the scaffold continues to expand, but can expand intoscaffolds of irregular shapes. In some embodiments, a weight ratio ofprecipitating agent to a polymer solution may range from about 0.05:1 toabout 3.5:1. In some embodiments, the weight ratio may range from about0.5:1 to 3:1. In yet other embodiments, the weight ratio may range inexcess of 3.5:1.

The expansion of the scaffold can be controlled by an amount ofprecipitating agent added. The expansion can alter the density and/orporosity of the scaffold relative to a scaffold where no precipitatingagent is used. In some embodiments, the scaffold has a thickness thatmay be at least about 2 times that of a scaffold where no precipitatingagent is used. In some embodiments, the scaffold has a thickness thatmay be at least about 3 times up to about 8 times. In yet anotherembodiment, the scaffold has a thickness that may be at least about 1order of magnitude greater than that of a scaffold where noprecipitating agent is used. In yet another embodiment, the scaffold hasa thickness that is greater than about 1 order of magnitude may beutilized.

In some embodiments, a density of the scaffold may have an inverselinear relationship with thickness of the expanded scaffold. Forexample, a scaffold made using a precipitating agent may have about 2times the thickness of a scaffold made without the use of aprecipitating agent, and about 50% of the density. In some embodiments,the density of a scaffold made using a precipitating agent may rangefrom about 90% to about 14% of the density of a scaffold made withoutusing a precipitating agent. In some embodiments, the density may rangefrom about 70% to about 14%. In other embodiments, the density may rangefrom about 50% to 14%. In yet other embodiments, the density may rangefrom about 40% to about 14%. In yet other embodiments, the density mayrange from about 29% to about 14%. In yet another embodiment, thedensity may be less than 14%. In yet another embodiment, a density ofabout 11% or less may be utilized. However, the relationship may notnecessarily be inverse linear in all cases, and may depend on the shapeof the mold used to make the scaffold.

The porosity of a scaffold made with a precipitating agent may begreater than that of a scaffold made without a precipitating agent. Insome embodiments, as density of the scaffold decreases, such as when anamount of precipitating agent added is increased, a fluid holdingcapacity, or porosity, can increase.

In some embodiments, surface topology of the scaffold and perhaps tosome extent scaffold thickness may be controlled based on how theprecipitating agent is added to the surface of the mixture. For example,a uniform, fine spray may yield a flat, dense surface. In otherembodiments, a slow, drop wise pipetting can result in a dimpled, fluffysurface. Slow to sudden submersion to various depths below the surfaceof the precipitating agent may result in scaffolds having differingdegrees of fluffy, sea foam-like surfaces and thicknesses. Whenprecipitating agent addition is carefully controlled by drop wiseaddition or a fine water spray, the dimensions of the resultingscaffolds can roughly approximate that of the molds in which they arecast. In some embodiments, a mold may have a thickness ranging fromabout 5 mm to about 30 mm. In some embodiments, a mold has a thicknessexceeding about 30 mm. In some embodiments, any suitable thickness ofmold can be utilized. In some embodiments, depending on a method ofscaffold precipitation the scaffold may exceed the thickness of the moldby one or more millimeters. The method may include adding at least oneof calcium phosphate and/or bioactive additive to the polymer solutionto form a mixture. Any suitable calcium phosphate or bioactive additivemay be utilized. The mixture may vary from a solution, or suspension, ora paste, depending on the application. For example, when calciumphosphate and/or bioactive glass particles are added, the mixture may bea suspension. Alternatively, if a soluble calcium phosphate and/orbioactive additive is used, the mixture may be a solution. In someembodiments, the mixture may be viscous, such as a paste, which can beobtained by controlling the amount of polymer solution relative tocalcium phosphate or bioactive additive, as well as the size of theparticle component of the paste. A paste may be utilized, for example,when coating the mixture on a substrate. However, solutions orsuspension of the mixture can also be used to coat substrates.

The amount of calcium phosphate and/or bioactive additive in the mixturemay vary depending on the application. In some embodiments, the amountof calcium phosphate may range from about 15 to about 50 wt %, based onthe total weight of the mixture. The total weight of the mixture may bethe summed weight of the individual components of the mixture, such ascalcium phosphate, bioactive additive, and polymer solution. In someembodiments, the amount of calcium phosphate may range from about 15 wt% to about 40 wt %, preferably about 20 wt % to about 40 wt %. In someembodiments, the mixture may be a paste, when calcium phosphate exceedsabout 20 wt %. An amount of a bioactive additive present in the mixturemay be dependent on the identity of the bioactive additive, and mayrange from about 0.001 wt % to about 80 wt % of the mixture. Forexample, a bioactive additive that is a growth factor may be present ina very low to low concentration, for example, ranging from about 0.001wt % to about 10 wt % of the mixture. For example, a bioactive additivethat is a structural component, such as collagen, may be present in ahigher concentration, for example, ranging from about 10 wt % to about80 wt % of the mixture.

The amount of polymer solution used relative to other components, suchas the calcium phosphate and/or the bioactive additive, may influencethe properties. For example, as shown in FIGS. 9A-9F, for a 10 wt %PCL-GAA solution, an amount of calcium phosphate of at least about 19 wt%, relative to the 10 wt % PCL-GAA solution, can produce a scaffold thatretains a shape of the mold in which the scaffold was made, and alsoretains the calcium phosphate. In contrast, below about 19 wt % in thisexemplary embodiment, the scaffold fails to retain the shape of themold, and at least some of the calcium phosphate.

The mixture may be formed in various ways. In some embodiments, thepolymer solution and at least one of the calcium phosphate or bioactiveadditive can be combined to form the mixture, and then the mixture maybe deposited on a substrate, in a mold, or in any suitable apparatus toshape a scaffold. In one embodiment, calcium phosphate is deposited in amold, and then the polymer solution is added to the mold to form themixture. In one embodiment, a substrate is dipped in the polymersolution, and then the coated substrate is dipped in, or otherwisecoated with, calcium phosphate. Similar methods of forming a mixture canbe applied for bioactive additives.

In some embodiments, the precipitating agent may be frozen in thecalcium phosphate and/or bioactive additive. For example, prior toadding the calcium phosphate and/or bioactive additive to the polymersolution, the calcium phosphate and/or bioactive additive may be soakedwith the precipitating agent, and frozen. The frozen calcium phosphateand/or bioactive additive may then be mixed with the polymer solution toform the mixture. The frozen calcium phosphate and/or bioactive additivewill thaw in the mixture and release the precipitating agent which canresult in precipitation of the polymer from the mixture to form ascaffold.

In some embodiments, the polymer solution may be frozen and then addedto the precipitating agent. In other embodiments, the mixture of thepolymer solution and calcium phosphate and/or the bioactive additive maybe frozen and then added to the precipitating agent. For example, thepolymer solution may be mixed with calcium phosphate and/or bioactiveadditive to form the mixture. The mixture may then be frozen, and thencontacted with the precipitating agent. As the frozen mixture thaws,precipitation of the polymer can occur to form a scaffold.

In one embodiment, a scaffold may be formed with a bioactive additivethat includes living cells. For example, such a scaffold may be used todeliver the living cells to tissues or implants. The living cells may befrozen in an appropriate freezing medium for cells. The frozen livingcells may be further encased in a frozen solution containing sodiumhydroxide or another base to form a frozen formulation. The frozenformulation could be submerged in the polymer solution to form themixture. As the base and polymer mix to reach pH neutrality, polymer canprecipitate around and encase the living cells to form a scaffold.

The method may comprise removing the solvent and/or precipitating agentfrom the scaffold. For example, after precipitation of the polymer thesolvent may be removed from the resulting scaffold by any suitablemethod. In some embodiments, the precipitating agent may be removed whenthe solvent is removed. Removal of the solvent and/or precipitatingagent may occur over any suitable time frame. This time frame maydepend, for example, on the size of the scaffold, amount of solventand/or precipitating agent used, the method of removal used, and thelike. In some embodiments, removal of the solvent and/or precipitatingagent may range up to about 24 hours. In some embodiments, removal ofthe solvent ranges from about 5 minutes to about 20 hours. In someembodiments, removal of the solvent ranges from about 5 minutes to about15 hours. In some embodiments, removal of the solvent ranges from about5 minutes to about 10 hours. In some embodiments, removal of the solventranges from about 5 minutes to about 5 hours. In some embodiments,removal of the solvent ranges from about 5 minutes to about 3 hours. Insome embodiments, removal of the solvent ranges from about 20 minutes toabout 1 hour. In yet other embodiments, removal of the solvent rangefrom about 15 minutes to about 30 minutes. In one embodiment, thesolvent may be removed by rinsing, which may result in dilution andremoval of the solvent. For example, the scaffold may be submerged inwater during the rinsing process. In one embodiment, the solvent may beremoved by evaporating the solvent under room temperature and ambientpressure conditions. In other embodiments, removal of the solvent may beaccelerated by exemplary processes, such as exposure to temperaturesbelow the melting point of the polymer of the scaffold, and also below atemperature that may inactivate, denature, or otherwise destroy anyother component of the scaffold, as well as ambient forced air, vacuumdrying, lyophilization, or other methods that can remove the solvent butdo not untowardly affect any other component of the scaffold. In oneexample, removal of a solvent in a PCL-containing scaffold may occur attemperatures below about 60° C. because PCL melts at about 60° C. Theabove-described methods for removal of the solvent may be used alone orin combination.

Further, for applications where the scaffold is used within the humanbody, sterilization may need to be performed. One common sterilizationmethod is gamma-beam irradiation. Polymers, such as PCL, and calciumphosphate are amenable to gamma-beam irradiation. In some embodiments,gamma-beam irradiation may affect the molecular weight and/or otheraspects of the chemical structure of the plastic comprising the scaffoldand thus the physical characteristics of the scaffold even when polymerssuch as PCL are used. Therefore, parameters of the method, such aspolymer concentration, molecular weight and the like, may be modified toaccount for a sterilization process.

The methods described herein can be further exemplified as discussed inthe Examples below.

Scaffold

The scaffolds formed herein have significant porosity, allowing them tosoak up and retain biological fluids like blood or bone marrow aspirate,which are often added to bone regeneration scaffolds prior toimplantation in patients. Also, their lower density, foam-likecomposition makes them easier to mold by hand and compress to fit into ahole or defect in bone. It also may be speculated that the lowerdensity, foam-like nature of the scaffolds described in the presentinvention may allow for their more rapid in vivo degradation andresorption in comparison with PCL-based scaffolds formed using othermethods.

The scaffolds described herein are in the form of a polymer matrix. Thescaffolds produced using the methods described herein may be differentin terms of final composition and physical properties as compared withscaffolds manufactured by other methods. The scaffolds described hereinmay have a foam-like appearance. The scaffolds may have a relativelyamorphous structure. The scaffolds may be non-crystalline. The densityand porosity of the scaffolds can be controlled by the methods describedherein to control a rate of bioresorption of the scaffold in vivo. Insome embodiments, the plastic component(s) of the scaffolds producedusing the methods discussed herein may not have a uniform or homogeneousfully interconnected pore structure. Moreover, in some embodiments, thescaffolds may have calcium phosphate and/or bioactive additive embeddedin the polymer matrix. In some embodiments, though embedded in thepolymer matrix, calcium phosphate and/or bioactive additive may retainat least some characteristic properties, such as porosity.

In some embodiments, the scaffold may be a polymer matrix, for example,made of only polymer and absent of additional materials embedded withinthe matrix and/or on the surface thereof, for example, as shown in FIGS.17A and 17B and discussed herein in Example 5-1. Such a scaffold may beutilized to hold open or expand a space prior to organ transplant, toinhibit or block tissue adhesions following surgery, or other medicalapplications that do not involve orthopedic applications, such as boneregeneration or bone replacement. Further, it is contemplated that suchscaffolds may be utilized in non-medical applications, such as forthermal or electrical insulation, packing materials, or the like.

In some embodiments, the scaffold includes the polymer matrix and atleast one of calcium phosphate or a bioactive additive embedded thereinand/or disposed on the surface thereof. The calcium phosphate may be anynumber of calcium phosphate materials as discussed herein. The bioactiveadditive may be any number of additives, such as collagen, bioactiveglass, and/or other additives as discussed herein. In some embodiments,the scaffold does not include any animal-derived materials, such ascollagen, living cells, or other animal-derived materials. Among otherreasons, the absence of animal-derived materials may preventimmunoreactivity, transmission of viral, bacterial, or prion infectionsand/or satisfy religious concerns in regards to animal-derivedmaterials, when such scaffolds are implanted as medical devices intohumans.

The scaffold may include a substrate. For example, the substrate maymodify the physical properties of the scaffold, such as a threedimensional substrate formed from a stiff polymer to improve mechanicalstrength of the scaffold. Exemplary substrates may include a mesh orscreen, such as those made of a metal, plastic, or fabric, a porouspolymer substrate, a bone suture anchor, which may be filamentous and/orcomprised of a textile, a porous metal implant, a tissue autograft orallograft or derivatives therefrom and/or living tissue.

The scaffold can have any suitable shape, for example, such as a sheet,a rectangle, a wedge, a cylinder, a square, a sphere, and the like, orthe shape may be irregular, such as sized to fit a particularapplication, such as a size and/or shape of a portion of bone beingfilled. A mold, or another apparatus, such as a substrate, may be usedto make a scaffold having any suitable shape.

Polymer

The polymer used in the methods described herein and scaffolds is notparticularly limiting, though polymers that are biocompatible and can bedispersed and/or dissolved in a solvent are preferred.

Any suitable resorbable biocompatible polymer may be used in accordancewith the present invention. Examples of suitable polymers include,without limitation, polycaprolactones (PCL), polyglycolides (PGA),polylactic acids (PLA), polyethylene, polypropylene, polystyrene,poly(D,L-lactic-co-glycolide) (PLGA), polyglycolic acid (PGA),poly-L-Lactic acid (PL-LA), polysulfones, polyolefins, polyvinyl alcohol(PVA), polyalkenoics, polyacrylic acids (PAA), polyesters, lower alkylcellulose ethers, methylcellulose, sodium carboxymethyl cellulose,hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethylcellulose, carboxymethyl cellulose, and mixturesthereof. In other embodiments, the biocompatible polymer may furthercontain gelatin and other suitable polymers described, for example, inU.S. Pat. Nos. 7,189,263; 7,534,451; 7,531,004; and 8,287915, which areincorporated herein by references in their entireties.

In some embodiments, the polymer may have a molecular weight (MW)ranging from about 3,000 g/mol to about 150,000 g/mol. In oneembodiment, the polymer may have a MW ranging from about 3,000 g/mol toabout 120,000 g/mol. In yet another embodiment, the polymer may have aMW ranging from about 3,000 g/mol to about 100,000 g/mol. In yet anotherembodiment, the polymer may have a MW ranging from about 3,000 g/mol toabout 80,000 g/mol. In yet another embodiment, the polymer may have amolecular weight (MW) ranging from about 14,000 g/mol to about 65,000g/mol. In one embodiment, the polymer may have a MW ranging from about40,000 g/mol to about 50,000 g/mol. In some embodiments, molecularweights above 150,000 g/mol can be utilized. In some embodiments, thepolymer may have a molecular weight (M_(n)) ranging from greater thanabout 45,000 g/mol up to about 80,000 g/mol. In some embodiments, M_(n)above 80,000 g/mol can be utilized. For example, different polymers anddifferent solvents can allow for polymers having higher MW to dissolveto form a polymer solution.

Calcium Phosphate

Various calcium phosphates are contemplated and include, for example,tetra-calcium phosphate, di-calcium phosphate, dicalcium phosphatedihydrous, dicalcium phosphate anhydrous, tri-calcium phosphate,mono-calcium phosphate, β-tricalcium phosphate, a-tricalcium phosphate,oxypatite, hydroxypatite, and mixtures thereof. However, for the sake ofbrevity, “calcium phosphate” includes any calcium salt known to thoseskilled in the art. The preparation of various forms of calciumphosphate for use in the present invention is described in U.S. Pat.Nos. 5,939,039, 6,383,519, 6,521,246, and 6,991,803, which areincorporated herein by reference in their entireties. Exemplary calciumphosphate products may include Vitoss® Bone Graft Substitutes, such asVitoss® micromorsels (1-2 mm) or Vitoss® subfines (<1 mm) (StrykerOrthobiologics, Malvern, Pa.).

Some embodiments of the scaffold may partially comprise materials, ormorsels, resulting from an oxidation-reduction reaction. These materialsmay be produced by methods comprising preparing an aqueous solution of ametal cation and at least one oxidizing agent. The solution is augmentedwith at least one soluble precursor anion oxidizable by said oxidizingagent to give rise to the precipitating agent oxoanion. Theoxidation-reduction reaction thus contemplated is conveniently initiatedby heating the solution under conditions of temperature and pressureeffective to give rise to said reaction. The oxidation-reductionreaction can cause at least one gaseous product to evolve and thedesired intermediate precursor mineral to precipitate from the solution.A reactive blend may be imbibed into a material that is capable ofabsorbing it to produce a porous mineral. It may be preferred that thematerial have significant porosity, be capable of absorbing significantamounts of the reactive blend via capillary action, and that the same besubstantially inert to reaction with the blend prior to its autologousoxidation-reduction reaction.

The intermediate precursor mineral thus prepared can either be used “asis” or can be treated in a number of ways. Thus, it may be heat-treatedgreater than about 800° C. or, preferably, greater than about 1100° C.in accordance with one or more paradigms to give rise to a preselectedcrystal structure or other preselected morphological structures therein.In some embodiments, the oxidizing agent is nitrate ion and the gaseousproduct is a nitrogen oxide, generically depicted as NO_(x)(g). Theprecursor mineral provided by the present methods be substantiallyhomogenous. As used in this context, substantially homogenous means thatthe porosity and pore size distribution throughout the precursor mineralis the same throughout.

In some embodiments, the intermediate precursor mineral may be anycalcium salt. Subsequent modest heat treatments convert the intermediatematerial to, e.g., novel monophasic calcium phosphate minerals or novelbiphasic β-tricalcium phosphate (β-TCP)+type-B, carbonated apatite(c-HAp) [β-Ca₃(PO₄)₂+Ca₅(PO₄)_(3-x)(CO₃)x(OH)] particulates. Morepreferably, the heat treatment converts the intermediate material to apredominantly β-TCP material.

In one embodiment, the calcium phosphate is □-TCP. In some embodiments,the calcium phosphate is porous. In some embodiment, the calciumphosphate contains micro-, meso-, and macroporosity. In some embodiment,the porosity of the calcium phosphate is interconnected. Macroporosityis characterized by pore diameters greater than about 100 μm and, insome embodiments, up to about 1000 μm to 2000 μm. Mesoporosity ischaracterized by pore diameters between about 10 μm and 100 μm, whilemicroporosity occurs when pores have diameters below about 10 μm. Insome embodiments, that macro-, meso-, and microporosity occursimultaneously and are interconnected in products. It is not necessaryto quantify each type of porosity to a high degree. Rather, personsskilled in the art can easily determine whether a material has each typeof porosity through examination, such as by mercury intrusionporosimetry, helium pycnometry or scanning electron microscopy. While itis certainly true that more than one or a few pores within the requisitesize range are needed in order to characterize a sample as having asubstantial degree of that particular form of porosity, no specificnumber or percentage is called for. Rather, a qualitative evaluation bypersons skilled in the art shall be used to determine macro-, meso-, andmicroporosity.

In some embodiments, the calcium phosphate is in the form of particlesor morsels and may contain a porous structure as described herein.

It will be appreciated that in some embodiments, the overall porosity ofthe calcium phosphate will be high. This characteristic is measured bypore volume, expressed as a percentage. Zero percent pore volume refersto a fully dense material, which, perforce, has no pores at all. Onehundred percent pore volume cannot meaningfully exist since the samewould refer to “all pores” or air. Persons skilled in the art understandthe concept of pore volume, however and can easily calculate and applyit. For example, pore volume may be determined in accordance withKingery, W. D., Introduction to Ceramics, Wiley Series on the Scienceand Technology of Materials, 1^(st) Ed., Hollowman, J. H., et al.(Eds.), Wiley & Sons, 1960, p. 409-417, which provides a formula fordetermination of porosity. Expressing porosity as a percentage yieldspore volume. The formula is: Pore Volume=(1−f_(p)) 100%, where f_(p) isfraction of theoretical density achieved.

Porosity can be measured by Helium Pycnometry. This procedure determinesthe density and true volume of a sample by measuring the pressure changeof helium in a calibrated volume. A sample of known weight anddimensions is placed in the pycnometer, which determines density andvolume. From the sample's mass, the pycnometer determines true densityand volume. From measured dimensions, apparent density and volume can bedetermined. Porosity of the sample is then calculated using (apparentvolume-measured volume)/apparent volume. Porosity and pore sizedistribution may also be measured by mercury intrusion porosimetry.

Pore volumes in excess of about 30% may be achieved while materialshaving pore volumes in excess of 50% or 60% may also be routinelyattainable. Some embodiments of the calcium phosphate may have porevolumes of at least about 70%. Some embodiments that may be preferredhave pore volumes in excess of about 75%, with 80% being still morepreferred. Some embodiments may have pore volume greater than about 90%,more preferably greater than about 92%. In some preferred cases, suchhigh pore volumes are attained while also attaining the presence ofmacro- meso- and microporosity as well as physical stability of thematerials produced.

It will be appreciated that the morsel size and content will be selectedbased on the desired application. For example, it may be necessary forthe scaffold to have one or more properties, such as elasticity,stiffness, tensile strength, and in vivo degradation rate. Morsel sizeand content of the morsels within the scaffold may be selected with oneor more of those properties in mind. The morsel size can range fromabout 100 μm to 2,000 μm, from about 200 μm to 900 μm, and from about212 μm to about 850 μm. Unless otherwise specified, morsel size as usedherein refers to the sieve size used to partition the calcium phosphatemorsels.

Due to the high porosity and broad pore size distribution (1 μm to 2000μm) of the morsels, the scaffold may be able to wick/soak/imbibematerials very quickly, and also be capable of retaining them. Materialsmay include a variety of fluids including blood, bone marrow aspirate,saline, antibiotics and proteins such as bone morphogenetic proteins(BMPs). Materials can also be imbibed with cells (e.g., fibroblasts,mesenchymal, stromal, marrow and stem cells), platelet rich plasma,other biological fluids, and any combination of the above. The scaffoldcan hold, maintain, and/or retain fluids once they are imbibed, allowingfor contained, localized delivery of imbibed fluids. This capability hasutility in cell-seeding, drug delivery, and delivery of biologicmolecules as well as in the application of bone tissue engineering,orthopedics, and carriers of pharmaceuticals.

Bioactive Additive

Various bioactive additives are contemplated, including natural andsynthetic bioactive additives. Exemplary bioactive additives may includebioactive glass, bone chips, demineralized bone chips or powder, livingcells, lyophilized bone marrow, collagen, other bioactive proteins orgrowth factors, biologics, peptides, glycosaminoglycans,anti-inflammatory compounds, antibiotics, anti-microbial elements, smallbiomolecules, active pharmaceutical ingredients, antibodies, and/ormixtures thereof.

The type of collagen used is not particularly limiting, and can includenative fibrous insoluble human, bovine, porcine, or synthetic collagen,soluble collagen, reconstituted collagen, or combinations thereof. Someembodiments of the scaffolds do not contain collagen.

“Bioactive glass” as used herein may be any alkali-containing ceramic,glass, glass-ceramic, or crystalline material that reacts as it comes incontact with physiologic fluids including, but not limited to, blood andserum, which leads to bone formation. In some embodiments, bioactiveglasses, when placed in physiologic fluids, form an apatite layer ontheir surface. Examples of bioactive glasses suitable for use aredescribed in U.S. Pat. No. 5,914,356, incorporated herein by reference.Suitable bioactive materials also include 45S5 glass and glass-ceramic,58S5 glass, S53P4 glass, apatite-wollastonite containing glass andglass-ceramic. The bioactive glass may be a glass-ceramic compositioncomprising heterogeneous particles having an irregular morphology andregions of combeite crystallites (“Combeite glass-ceramic” having thechemical composition Na₄Ca₃Si₆O₁₆(OH)₂). In some embodiments, thebioactive glass comprises about 5-50% by volume of regions of combeitecrystallites. Bioactive glasses suitable for use may be thosecompositions comprising calcium-phosphorous-sodium-silicate andcalcium-phosphorous-silicate. Such bioactive glasses include NovaBoneand NovaBone-AR, distributed by NovaBone Products, LLC, Alachua, Fla.Further bioactive glass compositions that may be suitable for use aredescribed in U.S. Pat. No. 6,709,744, which is incorporated herein byreference.

In some embodiments, resorption of bioactive glass particles of about150 μm or less occurs as silica as released within the apatite gellayer, while larger particles are eventually broken down by osteoclasts(Goasin, A. Bioactive Glass for Bone Replacement in CraniomaxillofacialReconstruction, Plastic and Reconstructive Surgery (2004) Vol. 114, No.2, pp. 590-593). The scaffold may provide appropriate bone growthindependent of the inclusion of bioactive glass. The role of thebioactive glass in the scaffold described herein may be stimulatory toosteoblasts, and as such, large particles of glass (>150 μm) may not benecessary, and thus the particles which are resorbed via dissolution arepreferred. However, all sizes of resorbable glass particles arecontemplated as suitable.

Particle size measurement is well known in the art. Unless otherwisespecified, particle size as used herein refers to the sieve size used topartition the glass particles. The bioactive glass particles may rangein size from about 20 μm to about 200 μm, or about 100 μm or less, orabout 150 μm or less, or about 30 um to about 200 um. The bioactiveglass particles may be bimodal in nature, with distinct particles in thesize range 32 μm-90 μm and particles in the size range 90 μm-150 μm. Thebioactive glass particles may be solid or may even be porous. In someembodiments, the bioactive glass is nonporous.

EXAMPLES Experimental Example 1: Scaffold Quality as a Function ofPolymer Concentration

Experimental Example 1 studies scaffold quality as a function of polymerconcentration in the polymer solution. Examples 1-1 through 1-3, theresults of which are depicted in FIGS. 8A-8C, were made using the samemethods, except different concentrations of polymer are used in thepolymer solution in each Example. Comparative Example 1-1, the result ofwhich is depicted in FIG. 8D, was made using the same methods, exceptusing a polymer concentration lower than that of Examples 1-1 through1-3.

A PCL filament-derived material for 3D printing (available fromMakerbot, M_(n)=about 50,000 g/mol) The PCL filament-derived materialwas cut into small pieces prior to further use. Though the Examplesherein used PCL filament-derived material, other types of PCL materialsmay be contemplated, for example, such as PCL pellets.

Example 1-1: Saturated PCL Solution

About 1.5 gram of PCL was added to about 10 g of glacial acetic acid(GAA) in a 50 ml plastic sample tube and the mixture was agitated overthe course of several hours by intermittently vortexing using a FisherScientific Vortex Mixer to form a PCL solution. Other forms of mixing orsample agitation may be contemplated to solubilize the PCL. The PCL-GAAsolution has a concentration of PCL greater than about 10 wt % and lessthan about 15 wt %. Concurrently, about 3.4 g of calcium phosphate(Vitoss® micromorsels, 1-2 mm, available from Stryker Orthobiologics)were placed into a mold (Aluminum, rectangular shaped, about 1.0 inchwide×4.0 inches long× 7/18 inches deep), where the mold was sprayed withcooking oil and excess oil wiped away with a kimwipe prior to adding thecalcium phosphate. About 12 g of the 10 wt %-15 wt % PCL-GAA solutionwas added to the mold containing the calcium phosphate, and the mixtureof PCL, GAA, and calcium phosphate was allowed to sit for about 5 min inthe mold. After which, about 1 to about 2 grams of water was added dropwise to the surface of the mixture, and the mixture was allowed toincubate for about 15 to about 30 minutes at room temperature andambient pressure conditions. After the incubation period, the mold wassubmerged to about 1 to about 2 mm below the surface of a water bath atabout room temperature and allowed to incubate for about 15 to about 30minutes. After the incubation period, and while submerged, the lateraledges of the scaffold were separated from the mold using a metalspatula. The mold and scaffold were further incubated for about 15 to 30minutes. After the incubation period the scaffold was removed from themold and air dried. The scaffold lost more than twice its weight duringdrying. The resulting scaffold is depicted in FIG. 8A.

Example 1-2: 10% PCL Solution

Example 1-2 is made using the same procedure described for Example 1-1,except about 1 g of PCL is added to about 10 g of GAA to form a 10 wt %PCL-GAA solution. The resulting scaffold is depicted in FIG. 8B.

Example 1-3: 7.5% PCL Solution

Example 1-3 is made using the same procedure described for Example 1-1,except about 0.75 g of PCL is added to about 10 g of GAA to form a 7.5wt % PCL_GAA solution. The resulting scaffold is depicted in FIG. 8C.

Comparative Example 1-1: 5.0% PCL Solution

Comparative Example 1-1 is made using the same procedure described forExample 1-1, except about 0.5 g of PCL is added to about 10 g of GAA toform a 5 wt % PCL-GAA solution. The resulting scaffold is depicted inFIG. 8D.

As shown in FIGS. 8A-8D, the scaffolds of Examples 1-1 through 1-3, andComparative Example 1-1 have bright white, styrofoam-like appearance,and in some cases exhibit a fluffy, sea-foam like surface. The scaffoldsroughly approximate the size of the mold from which the scaffolds werecast.

The concentration of PCL in the polymer solutions was found to affectthe physical characteristics of the resulting scaffold. Intactscaffolds, e.g., those that could easily be removed from the mold andretained a large fraction of the calcium phosphate added, were producedusing 10 wt %-15 wt %, 10 wt %, and 7.5 wt % PCL-GAA solutions as shownin FIGS. 8A-8C.

Scaffolds were assessed qualitatively for moldability by wetting themwith physiologic saline and hand kneading for one to two minutes.Scaffolds were qualitatively assessed to see if they resistedhand-molding and if so, to what extent; whether they could be torn apartby hand, and after molding if they remained cohesive; e.g., did thepolymer matrix remain intact and retain a large fraction of the calciumphosphate.

The scaffolds of Examples 1-1 and 1-2 could be torn apart by hand, wererelatively stiff and only slightly moldable, however the scaffoldsremained intact and retained most if not all of the calcium phosphate.The scaffold of Example 1-3 could be torn apart by hand, were absorbentand partially moldable, their polymer matrix remained intact but lostsome calcium phosphate during hand-kneading. The scaffold of ComparativeExample 1-1 could be torn apart by hand, however, the polymer matrix didnot remain intact upon de-molding and lost calcium phosphate easily uponde-molding.

Experimental Example 2: Scaffold Quality as a Function of Amount ofPolymer Solution

Experimental Example 2 studies scaffold quality as a function of amountof polymer solution. Examples 2-1 through 2-3, the results of which aredepicted in FIGS. 9A-9C, were made using the same methods, exceptdifferent amounts of polymer solution were used in each Example.Comparative Examples 2-1 through 2-3, the results of which are depictedin FIGS. 9D-9F, were made using the same methods, except using a polymerconcentration lower than that of Examples 2-1 through 2-3. Examples 2-1through 2-3 and Comparative Examples 2-1 through 2-3 were performedusing PCL filament-derived material as used in Experimental Example 1.

Example 2-1: 14 g Polymer Solution

A scaffold of Example 2-1 was prepared in the same manner as that ofExample 1-2, using a 10 wt % PCL-GAA solution, except using 14 g of 10wt % PCL-GAA solution. The resulting scaffold is depicted in FIG. 9A.

Example 2-2: 12 g Polymer Solution

A scaffold of Example 2-2 was prepared in the same manner as that ofExample 1-2. The resulting scaffold is depicted in FIG. 9B.

Example 2-3: 10 g Polymer Solution

A scaffold of Example 2-3 was prepared in the same manner as that ofExample 1-2, using a 10 wt % PCL-GAA solution, except using 10 g of the10 wt % PCL-GAA solution. The resulting scaffold is depicted in FIG. 9C.

Comparative Example 2-1: 8 g Polymer Solution

A scaffold of Comparative Example 2-1 was prepared in the same manner asthat of Example 1-2, using a 10 wt % PCL-GAA solution, except using 8 gof the 10 wt % PCL-GAA solution. The resulting scaffold is depicted inFIG. 9D.

Comparative Example 2-2: 6 g Polymer Solution

A scaffold of Comparative Example 2-2 was prepared in the same manner asthat of Example 1-2, using a 10 wt % PCL-GAA solution, except using 6 gof the 10 wt % PCL-GAA solution. The resulting scaffold is depicted inFIG. 9E.

Comparative Example 2-3: 4 g Polymer Solution

A scaffold of Comparative Example 2-3 was prepared in the same manner asthat of Example 1-2, using a 10 wt % PCL-GAA solution, except using 4 gof the 10 wt % PCL-GAA solution. The resulting scaffold is depicted inFIG. 9F.

The scaffolds of Examples 2-1 through 2-3, having about 10 g or greaterof 10 wt % PCL-GAA solution, exhibited robust and cohesive properties asshown in FIG. 9A-9C. However, the scaffolds of Comparative Examples 2-1through 2-3, having about 8 g or less of 10 wt % PCL-GAA solution, wereless robust and cohesive, and exhibited loss of at least calciumphosphate upon de-molding. In some cases, such as Comparative Example2-3, the scaffold additionally exhibited a loss of structure uponde-molding as shown in FIG. 9F.

Experimental Example 3: Scaffolds Using Other Polymers and Solvents

Experimental Example 3 studies biodegradable or non-biodegradablepolymers, other than those polymers used in Experimental Examples 1 and2, which may be used to create scaffolds.

Example 3-1: Poly(D,L-lactic-co-glycolide) in Acetone

A scaffold of Example 3-1 was made from a polymer solution where about1.7 g of poly (DL-lactic-co-glycolide) (50:50) (Available from SigmaAldrich Chemicals) was dispersed in about 12 g Acetone. The polymersolution was added to a mold which included about 3.4 g of calciumphosphate (Vitoss® micromorsels, available from Stryker Orthobiologics).The scaffold was prepared using water as a precipitating agent asdescribed in Experimental Example 1. The scaffold of Example 3-1exhibited poor integrity as shown in FIG. 10. It is possible thatincreasing the concentration of polymer in the polymer solution, similarto results obtained in Experimental Example 1, may yield a scaffoldhaving more desirable properties.

Experimental Example 4: Scaffold Including a Substrate

Scaffolds were prepared using various substrates, such as screens,porous polymer implants, three dimensional substrates, and bone sutureanchors.

Example 4-1: Scaffold Including a Screen

A paste was made from a mixture of a polymer solution (10 wt % PCL-GAA)and calcium phosphate (Vitoss® subfine, further sieved to obtain about250 pm average particle size) in a weight ratio of about 1.0:1.5-2.0.Using a metal spatula, the paste was smeared on both sides of astainless steel metal screen to cover about one half the surface area ofthe screen (316 Stainless, 50-100 mesh), and then immersed in water forabout 15 to 30 seconds to precipitate the PCL leaving a PCL-calciumphosphate layer adhered to, and covering both sides of the metal screenas shown in FIG. 11.

Example 4-2: Scaffold Including Porous Polymer Implants

As shown in FIG. 12A-12D, scaffolds can be formed using porous highdensity polyethylene (HDPE) sheets as substrates (FIGS. 12A-12C) or HDPEcranial implants (MedPor, FIG. 12D).

The scaffolds show in FIGS. 12A, 12B, and 12D were made using PCL as apolymer and calcium phosphate (Vitoss® particles, about 250 μm). In thescaffold depicted in FIG. 12C, bioactive glass was substituted in placeof calcium phosphate. The scaffolds of FIGS. 12A-12D were manufacturedby one of three methods: (1) immersing the materials in a coatingsolution including 10% PCL-GAA and one of calcium phosphate or bioactiveglass, (2) depositing the coating solution onto the substrates, forexample, using a brush or another deposition device, or (3) immersingthe substrates in a 10% PCL-GAA solution, and then dipping the wettedsubstrates in one of calcium phosphate or bioactive glass. In eachmethod, the coated substrates were then submerged under water for about15 to about 30 seconds to precipitate the polymer to form the scaffold.

In FIG. 12A, a porous HDPE sheet was dipped in a 10 wt % PCL-GAAsolution including calcium phosphate at the bottom edge, and thenprecipitated, resulting in a small, uneven area coated a scaffold. InFIG. 12B, a porous HDPE sheet was immersed in 10% PCL-GAA, and then thesurface of the porous HDPE sheet was pressed against a dry bed ofcalcium phosphate particles. It was then immersed in water toprecipitate the scaffold, resulting in the entire surface coated with,and white from, calcium phosphate. In FIG. 12C, a porous HDPE sheet wascoated similar to that of the sheet in FIG. 12B, except bioglass(Combeite, 90-150 microns) was used instead of calcium phosphate. Thesheet shown in FIG. 12C is completely coated with a PCL-bioglassscaffold but is not bright white like a scaffold made of PCL and calciumphosphate since the bioglass is clear to moderately cloudy buttranslucent, and not an opaque white.

Example 4-3: Scaffold Including Three-Dimensional Substrate

Example 4-3 uses a polylactic acid (PLA) wedge as a substrate as shownin FIG. 13A. The PLA has been 3D printed, and has a cage configurationwith a slotted base. A 10% PCL-GAA calcium phosphate (Vitoss®micromorsel suspension) was prepared in the same manner as described inExample 4-1, and packed into the interstices of the PLA wedge. Thepacked PLA wedge was submerged in a water bath for about 15 to about 30sec, and then air dried. The resulting scaffold comprised a stiff cageof PLA in which a matrix of PCL embedded with calcium phosphate wasstably incorporated throughout as shown in FIG. 13B.

Example 4-4: Scaffold Including Bone Suture Anchors

As shown in FIG. 14A, a scaffold can include a bone suture anchor as asubstrate. For example, one exemplary bone suture anchor is ICONIX™ bonesuture anchors, available from Stryker, Inc.

The scaffolds of Example 4-4 are prepared using a PCL-GAA-calciumphosphate mixture or a PCL-GAA-bioactive glass mixture, each used insame proportions as for other pastes described in Example 4-1, were usedto coat ICONIX bone suture anchors. The bone suture anchors are thendipped into the mixtures to coat the anchors with the mixture. Thecoated anchors are then submerged in a water bath for about 15 to about30 seconds to precipitate the polymer to form the scaffold, and then airdried.

As shown in FIGS. 14B-14D, a PCL-calcium phosphate (FIGS. 14B, 14D orPCL-bioactive glass scaffold (FIG. 14C) including a bone suture anchoras a substrate can be formed. At low magnification (FIGS. 14B-14D), thescaffolds appeared as thick white to white-gray coatings on the bonesuture anchor. As shown in FIG. 14D, the scaffolds have sufficientflexibility to be ‘bunched’, i.e., the bone suture anchor is subjectedto a manipulation that results in it having a different configuration.At high magnification (FIG. 15), scaffolds having bone suture anchorscoated with PCL-calcium phosphate exhibited a surface with a jaggedprofile. The jagged profile may be caused by the calcium phosphate,which can have a particulate form.

At low magnification (FIG. 14C, FIG. 16A) bioactive glass particles inscaffolds having bone suture anchors coated with PCL-bioactive glasswere too small to resolve. However, Energy-dispersive X-ray spectroscopy(EDAX) analysis (FIG. 16B, EDAX silicon map of FIG. 16A; FIG. 16C) ofthe PCL-bioactive glass scaffolds show elements consistent with both PCL(carbon and oxygen) and bioglass (calcium and silicon).

Experimental Example 5: Fine Structure and Atomic Composition ofScaffold

Experimental Example 5 examines fine structure and atomic composition ofscaffolds made with and without calcium phosphate by scanning electronmicroscopy (SEM) and EDAX, respectively.

Example 5-1: PCL Scaffold

Example 5-1 is made in the same manner as Example 1-3, except calciumphosphate is omitted. The scaffold of Example 5-1 had a sheet-like,porous appearance overall (FIG. 17A), in some areas appearing to becomprised of multiple layers, with some fibrous or fiber-like structures(FIG. 17B) that were of irregular diameters. Varying numbers of largeand small pores were also evident in many areas of the scaffold surface(FIGS. 17A, 17B). There was no evidence of a nano-fibrillar PCLcomposition as is characteristic of scaffolds fabricated byelectrospinning of solubilized PCL.

EDAX analysis (FIG. 18) of a region of the scaffold shown in FIG. 17Ashowed that the majority of the scaffold was comprised of carbon andoxygen, which was as expected for a scaffold made from a PCL solution.Some salts were present in small quantities, which is attributed toreagents used to make the scaffold. The presence of gold is due tosputter-coating the scaffold with gold during SEM sample preparation.

Example 5-2: PCL-Calcium Phosphate Scaffold

Example 5-2 is made in the same manner as Example 1-3. The scaffold ofExample 5-2 showed similar scaffold morphology to that of Example 5-1,except for the presence of calcium phosphate particles (Vitoss®micromorsels) were evident throughout as shown in FIG. 19. In addition,amorphous and ordered crystals appeared to be associated with both PCLand calcium phosphate.

EDAX analysis (FIGS. 20A, 20B) of regions of the scaffold depicted inFIG. 19 indicated the crystals comprised calcium phosphate. It isspeculated that the crystals may arise from acid solubilization (in GAA)of at least part of the calcium phosphate and then recrystallization ofthe calcium phosphate on the PCL. Calcium phosphate crystal depositionon the surface of the scaffold may be minimized if desired. For example,after precipitation of PCL, a copious water rinse and/or baseneutralization of GAA could be performed, optionally followed by rapiddrying.

Experimental Example 6: Scaffold Thickness as a Function of Amount ofPrecipitating Agent

Experimental Example 6 studies scaffold dimensions and quality as afunction of an amount of precipitating agent. Examples 6-1 through 6-5,the results of which are depicted in FIGS. 21A and 21B were made usingthe same methods, except different amounts of precipitating agent wereused in each Example. Comparative Example 6-1, the result of which isdepicted in FIG. 21B, was made using the same methods, except omitting aprecipitating agent. Scaffolds in Experimental Example 6 did not includea calcium phosphate and/or bioactive additive, such as Vitoss®micromorsels.

Example 6-1: Scaffold Prepared from 10% PCL-GAA Solution Using 1 g Water

A 10% PCL-GAA solution was prepared by the same methods as described inExample 1-2. About 2 g of the 10% PCL-GAA solution were added to a 25 mlborosilicate glass beaker (Kimax®, available from Kimble). About 1 g ofwater was added dropwise to the surface of the 10% PCL-GAA solution. Thewater was added over a period of less than one minute. The mixture wasallowed to incubate for about 15 to about 30 minutes at room temperatureand ambient pressure conditions. After the incubation period thescaffold was dried at room temperature and atmospheric conditions forabout 2 to 3 days and removed from the beaker. The scaffold is depictedin FIG. 21A and the thickness of the scaffold is depicted in comparisonto Examples 6-2 through 6 and Comparative Example 6-1 in FIG. 21B.

Examples 6-2 through 6-5: Scaffold Prepared from 10% PCL-GAA Solutionusing 2 g, 4 g, 6 g, and 10 g Water

Examples 6-2 through 6-5 are made using the same procedure described forExample 6-1, except about 2 g, about 4 g, about 6 g, and about 10 g ofwater, respectively, were added dropwise to the 10 wt % PCL-GAAsolution. The resulting scaffolds and thickness comparisons are depictedin FIGS. 21A, 21B.

Comparative Example 6-1: Scaffold Prepared from 10% PCL-GAA Solution andAir Drying

Comparative Example 6-1 is made using the same procedure described forExample 6-1, except water was omitted. The resulting scaffold andthickness comparison to the other Examples is depicted in FIGS. 21A,21B.

As shown in FIG. 21A, the scaffolds of Examples 6-2 through 6-5 have abright white, styrofoam-like appearance, and are porous. The scaffoldsexpand in height relative to the amount of water added up to about 6 g(Example 6-4). At higher amounts of water (8 g, not shown) or 10 g(Example 6-5) the scaffolds continue to expand to greater heights buttheir shapes become irregular. In contrast, Comparative Example 6-1,where water was not added, formed a thin, slightly flexible by manualmanipulation, wrinkled disk with little or no apparent porosity.

Comparative Example 7: Scaffold Prepared Using a Different Order ofProcess Steps

Comparative Example 7 is made using the same procedure described forExample 6-4, except about 6 g of water was added to the borosilicatebeaker, and 2 g of 10% PCL-GAA solution was added dropwise to the water.The resulting scaffold is depicted in FIG. 22 in comparison to thescaffold of Example 6-4. The scaffold of Comparative Example 6-2 ismushroom-shaped and has a non-homogenous density throughout thescaffold. The scaffold is also denser than that of Example 6-4.

Experimental Example 8: Scaffold Quality as a Function of MolecularWeight

Experimental Example 8 studies scaffold quality as a function ofmolecular weight of PCL. Examples 7-1 and Comparative Examples 8-1 and8-2 were made using the same methods, except different molecular weightsfor each polymer.

Example 8-1: Scaffold Prepared from 10% PCL-GAA Solution using Mn=80,000g/mol PCL

The scaffold of Example 7-1 was prepared by the same procedure asExample 1-2, except PCL pellets (Sigma-Aldrich Chemicals, Lot No.MKBV3325V) having Mn=80,000 g/mol were used in place of PCL filaments.

Comparative Example 8-1: Scaffold Prepared from 10% PCL-GAA Solutionusing Mn=45,000 g/mol PCL

The scaffold of Comparative Example 7-1 was prepared by the sameprocedure as Example 1-2, except PCL pellets (Sigma-Aldrich Chemicals,Lot No. MKBT6624V) having Mn=45,000 g/mol were used in place of PCLfilaments.

Comparative Example 8-2: Scaffold Prepared from 10% PCL-GAA Solutionusing Mn=14,000 g/mol PCL

The scaffold of Comparative Example 7-2 was prepared by the sameprocedure as Example 1-2, except PCL pellets (Sigma-Aldrich Chemicals,Lot No. MKBR890V) having Mn=14,000 g/mol were used in place of PCLfilaments.

Upon removing the scaffold of Example 7-1 from the mold, the scaffoldwas intact and robust. In contrast, the scaffold of Comparative Example7-1 was partially intact and insufficiently robust. The scaffold ofComparative Example 7-2 was not robust and fell apart upon removal fromthe mold.

It is to be understood that the disclosure set forth herein includes allpossible combinations of the particular features set forth above,whether specifically disclosed herein or not. For example, where aparticular feature is disclosed in the context of a particular aspect,arrangement, configuration, or embodiment, that feature can also beused, to the extent possible, in combination with and/or in the contextof other particular aspects, arrangements, configurations, andembodiments of the invention, and in the invention generally.

Furthermore, although the invention herein has been described withreference to particular features, it is to be understood that thesefeatures are merely illustrative of the principles and applications ofthe present invention. It is therefore to be understood that numerousmodifications, including changes in the sizes of the various featuresdescribed herein, may be made to the illustrative embodiments and thatother arrangements may be devised without departing from the spirit andscope of the present invention. In this regard, the present inventionencompasses numerous additional features in addition to those specificfeatures set forth in the claims below. Moreover, the foregoingdisclosure should be taken by way of illustration rather than by way oflimitation as the present invention is defined by the claims set forthbelow.

1. A method of forming a bioactive filamentary structure, comprising thesteps of: applying a polymer solution around a filamentary structure;after the polymer solution applying step, applying synthetic bone graftparticles to the polymer solution and around the filamentary structure;and after the synthetic bone graft particles applying step,precipitating a polymer from the polymer solution such that thesynthetic bone graft particles and the polymer coat the filamentarystructure.
 2. The method of claim 1, wherein the synthetic bone graftparticles are applied around the filamentary structure by placing thecoated filamentary structure into a container of synthetic bone graftparticles and then removing the filamentary structure from thecontainer.
 3. The method of claim 2, wherein the container of thesynthetic bone graft particles is shaken during placement of the coatedfilamentary structure into the container.
 4. The method of claim 1,wherein the synthetic bone graft particles include either one or both ofcalcium phosphate and a bioactive additive.
 5. The method of claim 1,wherein the polymer solution includes at least one solvent selected fromthe group consisting of glacial acetic acid (GAA), acetic acid, anisole,chloroform, methylene chloride, acetylchloride, 2,2,2 trifluoroethanol,trifluoroacetic acid, 1,2-Dochloroethane, and mixtures thereof.
 6. Themethod of claim 5, wherein the polymer solution comprisespolycaprolactone (PCL) and glacial acetic acid (GAA).
 7. The method ofclaim 1, wherein the polymer is precipitated from the polymer solutionby applying to the polymer solution a precipitating agent selected fromthe group consisting of sodium phosphate buffer, water, ethanol,1-propanol, isopropyl ether, 2-butanol, hexane, and mixtures thereof. 8.The method of claim 1, wherein the polymer solution is applied aroundthe filamentary structure by spraying the polymer solution around thefilamentary structure.
 9. The method of claim 1, wherein the step ofprecipitating the polymer from the polymer solution includes immersingthe polymer solution in a precipitating agent after applying both thesynthetic bone graft particles and the polymer solution around thefilamentary structure.
 10. The method of claim 1, wherein the step ofprecipitating the polymer from the polymer solution includes applying afirst buffer to the polymer solution after the polymer solution isapplied around the filamentary structure to at least partiallyneutralize the polymer solution.
 11. The method of claim 10, furthercomprising the step of applying a second buffer to the polymer solutionafter the first buffer is applied around the filamentary structure tofurther dilute the polymer solution.
 12. The method of claim 10, furthercomprising the step of drying the coated filamentary structure at leastafter the first buffer is applied to the polymer solution.
 13. Themethod of claim 12, further comprising the step of packaging the driedcoated filamentary structure disposed on an inserter.
 14. The method ofclaim 1, further comprising covering a portion of the filamentarystructure such that the bone graft particles do not coat the coveredportion of the filamentary structure during the synthetic bone graftparticles applying step.
 15. The method of claim 14, wherein thecovering step includes applying a mask over openings defined by thefilamentary structure.
 16. The method of claim 15, wherein the mask is atape or a film.
 17. The method of claim 1, wherein the bone graftparticles are wedged between fibers of the filamentary structure afterthe synthetic bone graft particles applying step.
 18. A method offorming a bioactive filamentary structure, comprising the steps of:mixing synthetic bone graft particles with a polymer solution to form ascaffold mixture; applying the scaffold mixture around a filamentarystructure; and precipitating a polymer from the polymer solution suchthat the synthetic bone graft particles and the polymer coat thefilamentary structure, wherein the bone graft particles are wedgedbetween fibers of the filamentary structure after the scaffold mixtureapplying step.
 19. The method of claim 18, further comprising covering aportion of the filamentary structure such that the bone graft particlesdo not coat the covered portion of the filamentary structure during thescaffold mixture applying step.
 20. The method of claim 18, furthercomprising any one or any combination of vibrating, translating androtating a container containing the bone graft particles and thefilamentary structure to wedge the bone graft particles between thefibers of the filamentary structure after the scaffold mixture applyingstep.
 21. A bioactive scaffold, comprising: a scaffold; synthetic bonegraft particles coating the scaffold; and a polymer coating thesynthetic bone graft particles such that at least some of the bone graftparticles extend at least partially through the polymer.